Sample records for expression cellular distribution

Glucose and other carbohydrates are transported into cells using members of a family of integral membrane glucose transporter (GLUT) molecules. To date 14 members of this family, also called the solute carrier 2A proteins have been identified which are divided on the basis of transport characteristics and sequence similarities into several families (Classes 1 to 3). The expression of these different receptor subtypes varies between different species, tissues and cellular subtypes and each has differential sensitivities to stimuli such as insulin. The liver is a contributor to metabolic carbohydrate homeostasis and is a major site for synthesis, storage and redistribution of carbohydrates. Situations in which the balance of glucose homeostasis is upset such as diabetes or the metabolic syndrome can lead metabolic disturbances that drive chronic organ damage and failure, confirming the importance of understanding the molecular regulation of hepatic glucose homeostasis. There is a considerable literature describing the expression and function of receptors that regulate glucose uptake and release by hepatocytes, the most import cells in glucose regulation and glycogen storage. However there is less appreciation of the roles of GLUTs expressed by non parenchymal cell types within the liver, all of which require carbohydrate to function. A better understanding of the detailed cellulardistribution of GLUTs in human liver tissue may shed light on mechanisms underlying disease pathogenesis. This review summarises the available literature on hepatocellular expression of GLUTs in health and disease and highlights areas where further investigation is required. PMID:23239915

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus linked to a range of acute and chronic neurological disorders affecting distinct regions of the brain. Unusually, HSV-1 entry into cells requires the interaction of viral proteins glycoprotein D (gD) and glycoprotein B (gB) with distinct cellular receptor proteins. Several different gD and gB receptors have been identified, including TNFRSF14/HVEM and PVRL1/nectin 1 as gD receptors and PILRA, MAG, and MYH9 as gB receptors. We investigated the expression of these receptor molecules in different areas of the adult and developing human brain using online transcriptome databases. Whereas all HSV-1 receptors showed distinct expression patterns in different brain areas, the Allan Brain Atlas (ABA) reported increased expression of both gD and gB receptors in the hippocampus. Specifically, for PVRL1, TNFRFS14, and MYH9, the differential z scores for hippocampal expression, a measure of relative levels of increased expression, rose to 2.9, 2.9, and 2.5, respectively, comparable to the z score for the archetypical hippocampus-enriched mineralocorticoid receptor (NR3C2, z = 3.1). These data were confirmed at the Human Brain Transcriptome (HBT) database, but HBT data indicate that MAG expression is also enriched in hippocampus. The HBT database allowed the developmental pattern of expression to be investigated; we report that all HSV1 receptors markedly increase in expression levels between gestation and the postnatal/adult periods. These results suggest that differential receptor expression levels of several HSV-1 gD and gB receptors in the adult hippocampus are likely to underlie the susceptibility of this brain region to HSV-1 infection.

Prostaglandins (PGs) play an important role in pulmonary physiology and various pathophysiological processes following infection. The initial step in the biosynthesis of PGs is regulated by two distinct cyclooxygenase enzymes, cyclooxygenase-1 (COX-1) and COX-2. The goal of this study was to investigate the pulmonary cellular localization and distribution of COX-1 and COX-2 in a neonatal lamb model following respiratory syncytial virus (RSV) and parainfluenza virus 3 (PI3) infection, organisms that also cause significant respiratory disease in children. No significant differences were seen in pulmonary COX-1 expression at various microanatomical locations following RSV or PI3 infection compared to controls. In contrast, COX-2 was upregulated following RSV and PI3 infection. Strong expression was restricted to bronchial and bronchiolar epithelial cells and macrophages, while minimal expression was present in the same microanatomical locations in the uninfected lungs. Other microanatomical locations in both the controls and the infected lungs lacked expression. This work suggests that during RSV or PI3 infection: (1) COX-1 cellularexpression is not altered, (2) COX-2 cellularexpression is upregulated in airway bronchiolar and bronchial epithelial cells and macrophages, (3) respiratory epithelium along with macrophages are important microanatomical compartments regulating the host inflammatory response during viral infection, and (4) COX-2 may be a potential target for RSV and PI3 therapy.

Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellulardistribution in spinal cord up to postnatal day P35 in control opossums (Monodelphis domestica) and in response to complete spinal transection (T10) at P7, when axonal growth through site of injury occurs, and P28 when this is no longer possible. Cords were collected 1 or 7 days after injury, with age-matched controls and segments rostral to lesion were studied. Following spinal injury ubiquitin levels (western blotting) appeared reduced compared to controls especially one day after injury at P28. In contrast, after injury mRNA expression (qRT-PCR) was slightly increased at P7 but decreased at P28. Changes in isoelectric point of separated ubiquitin indicated possible post-translational modifications. Cellulardistribution demonstrated a developmental shift between earliest (P8) and latest (P35) ages examined, from a predominantly cytoplasmic immunoreactivity to a nuclear expression; staining level and shift to nuclear staining was more pronounced following injury, except 7 days after transection at P28. After injury at P7 immunostaining increased in neurons and additionally in oligodendrocytes at P28. Mass spectrometry showed two ubiquitin bands; the heavier was identified as a fusion product, likely to be an ubiquitin precursor. Apparent changes in ubiquitin expression and cellulardistribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets. PMID:23626776

Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellulardistribution in spinal cord up to postnatal day P35 in control opossums (Monodelphis domestica) and in response to complete spinal transection (T10) at P7, when axonal growth through site of injury occurs, and P28 when this is no longer possible. Cords were collected 1 or 7 days after injury, with age-matched controls and segments rostral to lesion were studied. Following spinal injury ubiquitin levels (western blotting) appeared reduced compared to controls especially one day after injury at P28. In contrast, after injury mRNA expression (qRT-PCR) was slightly increased at P7 but decreased at P28. Changes in isoelectric point of separated ubiquitin indicated possible post-translational modifications. Cellulardistribution demonstrated a developmental shift between earliest (P8) and latest (P35) ages examined, from a predominantly cytoplasmic immunoreactivity to a nuclear expression; staining level and shift to nuclear staining was more pronounced following injury, except 7 days after transection at P28. After injury at P7 immunostaining increased in neurons and additionally in oligodendrocytes at P28. Mass spectrometry showed two ubiquitin bands; the heavier was identified as a fusion product, likely to be an ubiquitin precursor. Apparent changes in ubiquitin expression and cellulardistribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets.

Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellulardistribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellulardistribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells.

Although the expression of estrogen and progesterone receptors within porcine ovary and cumulus-oocyte complexes (COCs) is well recognized, still little information is known regarding expression of the progesterone receptor (PGR), PGR membrane component 1 (PGRMC1) and of estrogen-related receptors (ERRγ and ERRβ/γ) in separated cumulus cells in relation to real-time proliferation. In this study, a model of oocytes-separated cumulus cells was used to analyze the cell proliferation index and the expression PGR, PGRMC1 and of ERRγ and ERRβ/γ during 96-h cultivation in vitro using real-time quantitative PCR (qRT-PCR) and confocal microscopic observation. We found that PGR protein expression was increased at 0 h, compared with PGR protein expression after 96 h of culture (P < 0.001). The expression of PGRMC1, ERRγ and ERRβ/γ was unchanged. After using qRT-PCR we did not found statistical differences in expression of PGR, PGRMC1, ERRγ and ERRβ/γ during 96 h of cumulus cells in vitro culture (IVC). We supposed that the differential expression of the PGR protein at 0 h and after 96 h is related to a time-dependent down-regulation, which may activate a negative feedback. The distribution of PGR, PGRMC1 proteins may be linked with the translocation of receptors to the cytoplasm after the membrane binding of respective agonists and intra-cytoplasmic signal transduction. Furthermore, cumulus cells analyzed at 0 h were characterized by decreased proliferation index, whereas those after 96 h of culture revealed a significant increase of proliferation index, which may be associated with differentiation/luteinization of these cells during real-time proliferation.

Cumulus-oocyte-complexes (COCs) were collected from small (<3 mm), medium (3-5 mm), and large (>5 mm) porcine follicles, and the INHA and INHB expression and cellular localization were studied. Developmentally competent (BCB+) COCs were cultured for 44 h. Samples of mRNA were isolated before and after in vitro maturation (IVM) from oocytes collected from follicles of different size for RQ-PCR assay. The INHA and INHB protein distribution within the oocytes was observed by confocal microscopy. INHA mRNA expression was increased in oocytes from large compared to medium and small follicles before IVM (P < 0.001), and to oocytes of small follicles after IVM (P < 0.001). The INHB expression was not different before IVM, but the IHNB mRNA level was gradually higher in oocytes from large follicles after IVM (P < 0.01). INHA was not differently expressed before IVM; however, in large follicle oocytes the protein was distributed in the peripheral area of the cytoplasm; in oocytes from small follicles it was in the entire cytoplasm. After IVM, INHA was strongly expressed in oocytes from small follicles and distributed particularly in the zona pellucida (ZP). Similarly and both before and after IVM, INHB protein was highly expressed in small follicle oocytes and within the cytoplasm. In summary, INHs can be recognized as a marker of porcine oocyte quality.

Osmoprotective genes are tonicity-activated genes involved in cellular osmoadaptation to hypertonicity and considered to be regulated by a specific transcription factor called tonicity-responsive enhancer-binding protein (TonEBP). In the brain we had previously established that TonEBP was expressed and tonicity-induced in neurons only. Here we have compared in various brain regions of rats subjected to systemic hypertonicity, the cellularexpression of TonEBP through immunocytochemistry and the cellularexpression of osmoprotective genes, namely aldose reductase (AR), sodium-dependent myo-inositol transporter (SMIT), betaine/GABA transporter (BGT1) and taurine transporter (TauT), by in situ hybridization using non-radioactive digoxigenin-labeled riboprobes. In neurons where TonEBP was strongly tonicity-induced, AR-mRNA labeling was strongly increased in some subsets (e.g. hippocampus pyramidal cells, cerebellar Purkinje cells and neurons of the hypothalamic magnocellular nuclei) but remained undetectable in some other subsets (e.g. neurons in cerebral cortex). Tonicity-induced AR-mRNA labeling was observed only several hours after the tonicity-induced expression of TonEBP. SMIT-mRNA labeling was tonicity-induced as densely and evenly distributed dots in neuron poor regions (e.g. cerebral cortex layer I and hippocampus stratum lacunosum-moleculare). The tonicity-induced expression of SMIT-mRNA may thus occur in non-neuronal cells, presumably astrocytes, where TonEBP is neither significantly expressed, nor tonicity-induced. In neurons showing a strong tonicity-induced expression of TonEBP, no SMIT-mRNA labeling was observed. BGT1-mRNA and TauT-mRNA labeling could not be detected, even after systemic hypertonicity. The present work reveals large discrepancies between the cellulardistribution of the tonicity-induced expression of osmoprotective genes and that of their regulatory transactivator TonEBP. Depending on the cell subsets and the osmoprotective genes, Ton

Granulosa cells (GCs) play an important role during follicle growth and development in preovulatory stage. Moreover, the proteins such as connexins are responsible for formation of protein channel between follicular-cumulus cells and oocyte. This study was aimed to investigate the role of connexin expression in porcine GCs in relation to their cellulardistribution and real-time cell proliferation. In the present study, porcine GCs were isolated from the follicles of puberal gilts and then cultured in a real-time cellular analyzer (RTCA) system for 168 h. The expression levels of connexins (Cxs) Cx36, Cx37, Cx40 and Cx43 mRNA were measured by RQ-PCR analysis, and differences in the expression and distribution of Cx30, Cx31, Cx37, Cx43 and Cx45 proteins were analyzed by confocal microscopic visualization. We found higher level of Cx36, Cx37, and Cx43 mRNA expression in GCs at recovery (at 0 h of in vitro culture, IVC) compared to all analyzed time periods of IVC (24, 48, 72, 96, 120, 144 and 168 h; P<0.001). On the other hand, the expression level of Cx40 transcripts was higher after 24 h of IVC compared to 0 h and the other times of IVC (P<0.001). Similarly to mRNAs, the expression levels of Cx31, Cx37 and Cx45 proteins were higher before (0 h) compared to after 168 h of IVC. The expression of Cx30 and Cx43, however, did not vary between the groups. In all, the proteins were distributed throughout the cell membrane rather than in the cytoplasm both before and after IVC. After 24 h of IVC, we observed a significant increase in the proliferation of GCs (log phase). We found differences in the proliferation index between 72-96 and 96- 140 h within the same population of GCs. In conclusion, the decrease in the expression of Cx mRNAs and proteins following IVC could be associated with a breakdown in gap-junction connections (GJCs), and leads to the decreased of their activity, which may be a reason of non-functional existence of connexon in follicular granulosa cells

In a previous work performed on cerebral cortex and hippocampus we reported that tonicity-responsive enhancer binding protein (TonEBP), originally identified as a transactivator of osmoprotective genes involved in osmoadaptation of renal cells, was induced in neurons only, but to varying levels, following acute systemic hypertonicity. Whether or not this cellular specificity reflected a unique ability of neurons or a differential time course among brain cells for tonicity-induction of TonEBP was investigated throughout the brain in this study by subjecting the animals to prolonged systemic hypertonicity. In normal rats, TonEBP immunolabeling and TonEBP-mRNA in situ hybridization labeling showed a widespread, uneven and parallel distribution. TonEBP was expressed primarily in the cell nuclei of neurons, where it was heterogeneously distributed in a nucleoplasmic and a granular pool. In rats subjected to prolonged systemic hypertonicity, TonEBP labeling increased in the cell nuclei of neurons only. The tonicity-induced expression of TonEBP for a given cell group of neurons was rather uniform but varied greatly among neuronal cell groups and was positively correlated with the average size of the cell nuclei, as determined by quantitative analysis of digitized images. The detailed distribution of tonicity-induced expression of TonEBP is reported throughout the brain. In normal rats, a very minor proportion of non-neuronal cells, identified as a subset of astrocytes and possibly oligodendrocytes, showed faint nuclear immunolabeling, which however did not increase in hypertonic animals. Ependymocytes, capillary endothelial cells, and microglial cells showed no TonEBP labeling, even in hypertonic animals. Altogether our data indicate that neurons, albeit possibly to a varying extent, are the only brain cells able to use TonEBP-mediated processes for adaptation to a systemic hyperosmotic unbalance.

Wealth distribution of a country is a complicate system. A model, which is based on the Epstein & Axtell's "Sugars cape" model, is presented in Netlogo. The model considers the income, age, working opportunity and salary as control variables. There are still other variables should be considered while an artificial society is established. In this study, a more complicate cellular automata model for wealth distribution model is proposed. The effects of social welfare, tax, economical investment and inheritance are considered and simulated. According to the cellular automata simulation for wealth distribution, we will have a deep insight of financial policy of the government.

Epstein-Barr virus (EBV) is associated with several types of lymphomas and epithelial tumors including Burkitt's lymphoma (BL), HIV-associated lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. EBV nuclear antigen 1 (EBNA1) is expressed in all EBV associated tumors and is required for latency and transformation. EBNA1 initiates latent viral replication in B cells, maintains the viral genome copy number, and regulates transcription of other EBV-encoded latent genes. These activities are mediated through the ability of EBNA1 to bind viral-DNA. To further elucidate the role of EBNA1 in the host cell, we have examined the effect of EBNA1 on cellular gene expression by microarray analysis using the B cell BJAB and the epithelial 293 cell lines transfected with EBNA1. Analysis of the data revealed distinct profiles of cellular gene changes in BJAB and 293 cell lines. Subsequently, chromatin immune-precipitation revealed a direct binding of EBNA1 to cellular promoters. We have correlated EBNA1 bound promoters with changes in gene expression. Sequence analysis of the 100 promoters most enriched revealed a DNA motif that differs from the EBNA1 binding site in the EBV genome.

Cellular oncogenes have been implicated in the induction of malignant transformation in some model systems in vitro and may be related to malignancies in vivo in some vertebrate species. This article describes a study of the expression of 15 cellular oncogenes in fresh human tumors from 54 patients, representing 20 different tumor types. More than one cellular oncogene was transcriptionally active in all of the tumors examined. In 14 patients it was possible to study normal and malignant tissue from the same organ. In many of these patients, the transcriptional activity of certain oncogenes was greater in the malignant than the normal tissue. The cellular fes (feline sarcoma) oncogene, not previously known to be transcribed in mammalian tissue, was found to be active in lung and hematopoietic malignancies.

Autophagy is essential for the maintenance of cellular metabolism. Once autophagy is induced in cells, the isolation membrane forms a so-called phagophore. The endoplasmic reticulum (ER) is one of several candidates for the membrane source for phagophores. Recently, LC3-positive isolation membranes were found to emerge from a DFCP1 (double FYVE domain-containing protein)-positive, ER-associated compartment called the omegasome. Although the GFP-tagged DFCP1 protein has been examined in cultured cells, little is known about the precise cellular and tissue distribution of this endogenous protein. To determine the expression of the endogenous DFCP1 protein, we produced antibodies specific to mouse DFCP1 protein. The antibody recognized both human and mouse DFCP1 proteins, both of which have molecular masses of approximately 87 kDa. In HeLa cells under normal conditions, immunoreactivity for DFCP1 was found dotted or tubular along Tom20-positive filamentous mitochondria and was only partially co-localized in the ER or Golgi apparatus. Moreover, under starved conditions, distinct DFCP1-positive structures became more dotted and scattered in the cytoplasm, while one part of the LC3-positive autophagosomes were immunopositive for DFCP1. These results indicate that an antibody raised against DFCP1 could be a useful tool in explaining the mechanism of phagophore formation from omegasome compartments.

We previously reported that tocotrienols acted as more potent inhibitors against selenium deficiency-induced cell death than the corresponding tocopherol isoforms (J. Biol. Chem. 2003;278:39428-39434). In the present study, we first compared the differences in the cellular uptake between alpha-tocopherol (alpha-Toc) and alpha-tocotrienol (alpha-Toc-3). The initial rate of cellular uptake of alpha-Toc-3 was 70-fold higher than that of alpha-Toc. Subcellular fractionation analysis of alpha-Toc-3 and alpha-Toc-fortified cells showed similar cellulardistribution of these antioxidants, which was directly proportional to the lipid distribution. The cells containing similar amounts of alpha-Toc-3 and alpha-Toc showed similar resistance against the oxidative stress caused by peroxides. These results suggest that the apparent higher cytoprotective effect of alpha-Toc-3 than alpha-Toc is primarily ascribed to its higher cellular uptake.

Summary The integration of synthetic and cell-free biology has made tremendous strides towards creating artificial cellular nanosystems using concepts from solution-based chemistry: only the concentrations of reacting species modulate gene expression rates. However, it is known that macromolecular crowding, a key feature of natural cells, can dramatically influence biochemical kinetics by volume exclusion effects that reduce diffusion rates and enhance binding rates of macromolecules. Here, we demonstrate that macromolecular crowding can increase the robustness of gene expression through integrating synthetic cellular components of biological circuits and artificial cellular nanosystems. In addition, we reveal how ubiquitous cellular modules, including genetic components, a negative feedback loop, and the size of crowding molecules, can fine tune gene circuit response to molecular crowding. By bridging a key gap between artificial and living cells, our work has implications for efficient and robust control of both synthetic and natural cellular circuits. PMID:23851358

The integration of synthetic and cell-free biology has made tremendous strides towards creating artificial cellular nanosystems using concepts from solution-based chemistry, where only the concentrations of reacting species modulate gene expression rates. However, it is known that macromolecular crowding, a key feature in natural cells, can dramatically influence biochemical kinetics via volume exclusion effects, which reduce diffusion rates and enhance binding rates of macromolecules. Here, we demonstrate that macromolecular crowding can increase the robustness of gene expression by integrating synthetic cellular components of biological circuits and artificial cellular nanosystems. Furthermore, we reveal how ubiquitous cellular modules, including genetic components, a negative feedback loop and the size of the crowding molecules can fine-tune gene circuit response to molecular crowding. By bridging a key gap between artificial and living cells, our work has implications for efficient and robust control of both synthetic and natural cellular circuits.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a RNA binding protein that plays important role in the biogenesis of mRNA, such as alternative splicing and mRNA stability. We have previously demonstrated that hnRNP A1 has diminished protein levels and shows cytoplasmic accumulation in senescent human diploid fibroblasts. Recent reports showed that p38 MAP kinase (p38 MAPK), a member of the MAP kinase family is necessary and sufficient for the cytoplasmic accumulation of hnRNP A1 by stress stimuli such as osmotic shock. p38 MAP kinase has been shown to be involved in cell proliferation and the induction of senescence in response to extracellular stimuli. However, the relationship between hnRNP A1 and p38 MAPK and the roles of hnRNP A1 in cellular senescence have not yet been elucidated. Here we show that hnRNP A1 forms a complex with phospho-p38 MAPK in vivo. Inhibition of p38 MAPK activity with SB203580 elevated hnRNP A1 protein levels and prohibited the cytoplasmic accumulation of the protein, but not hnRNP A2, in senescent cells. The phosphorylation level of hnRNP A1 was elevated in senescent cells. Reduction of hnRNP A1 and A2 levels by siRNA transfection induced a senescence-like morphology and elevated the level of F-actin, a marker of senescence. These results suggest that the expression levels and subcellular distribution of hnRNP A1 are regulated in a p38 MAPK-dependent manner, probably via its phosphorylation. Our results also suggest that hnRNP A2 in addition to hnRNP A1 may play a role in establishing the senescence phenotype. PMID:19430204

The objective of this study was to analyze cellular localization and expression levels of oncologic relevant members of the S100 family in common oral lesions.Biopsies of various oral lesions were analyzed. S100A4 showed a higher expression rate in leukoplakias and oral squamous cell carcinomas. Transcript levels of S100A8 and S100A9 were significantly decreased in malignant OSCCs. A correlation could be drawn between the expression levels of these genes and the pathological characteristics of the investigated lesions. S100A4, A8, and A9 proteins represent promising marker genes to evaluate the risk potential of suspicious oral lesions in molecular pathology.

Cellular electrical activity is the result of a highly complex processes that involve the activation of ion channel proteins. Ion channels make pores on cell membranes that rapidly transit between conductive and non-conductive states, allowing different ions to flow down their electrochemical gradients across cell membranes. In the case of neuronal cells, ion channel activity orchestrates action potentials traveling through axons, enabling electrical communication between cells in distant parts of the body. Somatic sensation -our ability to feel touch, temperature and noxious stimuli- require ion channels able to sense and respond to our peripheral environment. Sensory integration involves the summing of various environmental cues and their conversion into electrical signals. Members of the Transient Receptor Potential (TRP) family of ion channels have emerged as important mediators of both cellular sensing and sensory integration. The regulation of the spatial and temporal distribution of membrane receptors is recognized as an important mechanism for controlling the magnitude of the cellular response and the time scale on which cellular signaling occurs. Several studies have shown that this mechanism is also used by TRP channels to modulate cellular response and ultimately fulfill their physiological function as sensors. However, the inner-working of this mode of control for TRP channels remains poorly understood. The question of whether TRPs intrinsically regulate their own vesicular trafficking or weather the dynamic regulation of TRP channel residence on the cell surface is caused by extrinsic changes in the rates of vesicle insertion or retrieval remain open. This review will examine the evidence that sub-cellular redistribution of TRP channels plays an important role in regulating their activity and explore the mechanisms that control the trafficking of vesicles containing TRP channels.

The assessment of radiotoxicity for heterogeneously distributed tritium should be based on the subcellular dose and relative biological effectiveness (RBE) for cell nucleus. In the present work, geometry-dependent absorbed dose and RBE were calculated using Monte Carlo codes for tritium in the cell, cell surface, cytoplasm, or cell nucleus. Penelope (PENetration and Energy LOss of Positrins and Electrons) code was used to calculate the geometry-dependent absorbed dose, lineal energy, and electron fluence spectrum. RBE for the intestinal crypt regeneration was calculated using a lineal energy-dependent biological weighting function. RBE for the induction of DNA double strand breaks was estimated using a nucleotide-level map for clustered DNA lesions of the Monte Carlo damage simulation (MCDS) code. For a typical cell of 10 μm radius and 5 μm nuclear radius, tritium in the cell nucleus resulted in much higher RBE-weighted absorbed dose than tritium distributed uniformly. Conversely, tritium distributed on the cell surface led to trivial RBE-weighted absorbed dose due to irradiation geometry and great attenuation of beta particles in the cytoplasm. For tritium uniformly distributed in the cell, the RBE-weighted absorbed dose was larger compared to tritium uniformly distributed in the tissue. Cellular- and micro-dosimetry models were developed for the assessment of heterogeneously distributed tritium.

Stably Expressed Genes (SEGs) whose expression varies within a narrow range may be involved in core cellular processes necessary for basic functions. To identify such genes, we re-analyzed existing RNA-Seq gene expression profiles across 11 organs at 4 developmental stages (from immature to old age) in both sexes of F344 rats (n = 4/group; 320 samples). Expression changes (calculated as the maximum expression / minimum expression for each gene) of >19000 genes across organs, ages, and sexes ranged from 2.35 to >109-fold, with a median of 165-fold. The expression of 278 SEGs was found to vary ≤4-fold and these genes were significantly involved in protein catabolism (proteasome and ubiquitination), RNA transport, protein processing, and the spliceosome. Such stability of expression was further validated in human samples where the expression variability of the homologous human SEGs was significantly lower than that of other genes in the human genome. It was also found that the homologous human SEGs were generally less subject to non-synonymous mutation than other genes, as would be expected of stably expressed genes. We also found that knockout of SEG homologs in mouse models was more likely to cause complete preweaning lethality than non-SEG homologs, corroborating the fundamental roles played by SEGs in biological development. Such stably expressed genes and pathways across life-stages suggest that tight control of these processes is important in basic cellular functions and that perturbation by endogenous (e.g., genetics) or exogenous agents (e.g., drugs, environmental factors) may cause serious adverse effects.

Stably Expressed Genes (SEGs) whose expression varies within a narrow range may be involved in core cellular processes necessary for basic functions. To identify such genes, we re-analyzed existing RNA-Seq gene expression profiles across 11 organs at 4 developmental stages (from immature to old age) in both sexes of F344 rats (n = 4/group; 320 samples). Expression changes (calculated as the maximum expression / minimum expression for each gene) of >19000 genes across organs, ages, and sexes ranged from 2.35 to >109-fold, with a median of 165-fold. The expression of 278 SEGs was found to vary ≤4-fold and these genes were significantly involved in protein catabolism (proteasome and ubiquitination), RNA transport, protein processing, and the spliceosome. Such stability of expression was further validated in human samples where the expression variability of the homologous human SEGs was significantly lower than that of other genes in the human genome. It was also found that the homologous human SEGs were generally less subject to non-synonymous mutation than other genes, as would be expected of stably expressed genes. We also found that knockout of SEG homologs in mouse models was more likely to cause complete preweaning lethality than non-SEG homologs, corroborating the fundamental roles played by SEGs in biological development. Such stably expressed genes and pathways across life-stages suggest that tight control of these processes is important in basic cellular functions and that perturbation by endogenous (e.g., genetics) or exogenous agents (e.g., drugs, environmental factors) may cause serious adverse effects. PMID:28125669

We took advantage of one of the main possibilities of ion microscopy, ie isotopic analysis, to study the cellulardistribution of molecules labelled either with carbon 14 or with stable isotopes of low natural abundance such as nitrogen 15 and deuterium. The surface of the sample is bombarded with an ion beam (O2+, Cs+ etc). Secondary ions emitted from the sample are filtered by a mass spectrometer and the distribution of the labelling isotope is recorded. In this way, we obtained images showing the characteristic distribution of 14C-thymidine and D-arginine in human fibroblasts, and of 15N-adenine in organotypic cultures of human breast cancer cells. The spatial resolution on the acquired images was close to 0.1 micron when using the UPS-ONERA ion microprobe. The sensitivity of the method for detecting carbon 14 is far greater than that of autoradiography and the technique is both fast and quantitative. On the other hand, the capacity of ion microscopy for studying the tissular distribution of molecules labelled with stable isotopes, opens the way for biological and pharmacological tracer studies of human diseases.

Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body.

The expression and function of TRPV1 is influenced by its interaction with cellular proteins. Here, we identify whirlin, a cytoskeletal PDZ-scaffold protein implicated in hearing, vision and mechanosensory transduction, as an interacting partner of TRPV1. Whirlin associates with TRPV1 in cell lines and in primary cultures of rat nociceptors. Whirlin is expressed in 55% of mouse sensory C-fibers, including peptidergic and non-peptidergic nociceptors, and co-localizes with TRPV1 in 70% of them. Heterologous expression of Whirlin increased TRPV1 protein expression and trafficking to the plasma membrane, and promoted receptor clustering. Silencing Whirlin expression with siRNA or blocking protein translation resulted in a concomitant degradation of TRPV1 that could be prevented by inhibiting the proteasome. The degradation kinetics of TRPV1 upon arresting protein translation mirrored that of Whirlin in cells co-expressing both proteins, suggesting a parallel degradation mechanism. Noteworthy, Whirlin expression significantly reduced TRPV1 degradation induced by prolonged exposure to capsaicin. Thus, our findings indicate that Whirlin and TRPV1 are associated in a subset of nociceptors and that TRPV1 protein stability is increased through the interaction with the cytoskeletal scaffold protein. Our results suggest that the Whirlin-TRPV1 complex may represent a novel molecular target and its pharmacological disruption might be a therapeutic strategy for the treatment of peripheral TRPV1-mediated disorders. PMID:26516054

The association between tissue factor (TF) expression and increased rate of tumour metastasis is well established. In this study, we have examined the hypothesis that the expression of TF by disseminated tumour cells confers protection against immune recognition and cytotoxicity. A hybrid EGFP-TF protein was expressed in HT29 colon carcinoma and K562 lymphoblast cell lines. To assess the cytotoxic activity against tumour cells over-expressing TF, a novel method was used, based on the direct measurement of fluorescently labelled HT29 or K562 target cells. Upon challenge with peripheral blood mononuclear cells (PBMC), tumour cells expressing TF partially evaded cellular cytotoxicity (Delta=15-40% reduction in cytotoxicity). Moreover, the influence of TF was not primarily dependent on its procoagulant function, although the inclusion of 20% (v/v) plasma did lower the rate of cytotoxicity against untransfected cells. However, expression of a truncated form of TF, devoid of the cytoplasmic domain, did not mediate any degree of inhibition of cytotoxicity, suggesting that the protective function of TF is principally due to this domain. We conclude that TF can promote immune evasion in tumour cells expressing this protein leading to increased survival and therefore metastatic rate in such cells.

Although often overlooked, passive mechanisms can lead to significant accumulation or restriction of drugs to intracellular sites of drug action. These mechanisms include lipoidal diffusion of ionized species and pH partitioning according to the electrochemical potential and to pH gradients that exist across subcellular compartments, respectively. These mechanisms are increasingly being exploited in the design of safe and effective drugs for the treatment of a wide variety of diseases. In this work, the authors review these efforts and the associated passive mechanisms of cellular drug permeation. A generic mathematical model of the cell is provided and used to illustrate concepts relevant to steady-state intracellular distribution. Finally, the authors review methods for estimating determinant parameters and measuring the net effect at the level of unbound intracellular drug concentrations.

The translocase of the mitochondrial outer membrane (TOM) complex is the main import pore for nuclear-encoded proteins into mitochondria, yet little is known about its spatial distribution within the outer membrane. Super-resolution stimulated emission depletion microscopy was used to determine quantitatively the nanoscale distribution of Tom20, a subunit of the TOM complex, in more than 1,000 cells. We demonstrate that Tom20 is located in clusters whose nanoscale distribution is finely adjusted to the cellular growth conditions as well as to the specific position of a cell within a microcolony. The density of the clusters correlates to the mitochondrial membrane potential. The distributions of clusters of Tom20 and of Tom22 follow an inner-cellular gradient from the perinuclear to the peripheral mitochondria. We conclude that the nanoscale distribution of the TOM complex is finely adjusted to the cellular conditions, resulting in distribution gradients both within single cells and between adjacent cells.

Nowadays, there is a growing interest in providing internet to mobile users. For instance, NTT DoCoMo in Japan deploys an important mobile phone network with that offers the Internet service, named 'i-mode', to more than 17 million subscribers. Internet traffic measurements show that the session duration of Call Holding Time (CHT) has probability distributions with heavy-tails, which tells us that they depart significantly from the traffic statistics of traditional voice services. In this environment, it is particularly important to know the number of handovers during a call for a network designer to make an appropriate dimensioning of virtual circuits for a wireless cell. The handover traffic has a direct impact on the Quality of Service (QoS); e.g. the service disruption due to the handover failure may significantly degrade the specified QoS of time-constrained services. In this paper, we first study the random behavior of the number of handovers during a call, where we assume that the CHT are Pareto distributed (heavy-tail distribution), and the Cell Residence Times (CRT) are exponentially distributed. Our approach is based on renewal theory arguments. We present closed-form formulae for the probability mass function (pmf) of the number of handovers during a Pareto distributed CHT, and obtain the probability of call completion as well as handover rates. Most of the formulae are expressed in terms of the Whittaker's function. We compare the Pareto case with cases of $k(subscript Erlang and hyperexponential distributions for the CHT.

Biological cells are extremely sensitive to temperature. What is the mechanism? We compute the thermal stabilities of the whole proteomes of Escherichia coli, yeast, and Caenorhabditis elegans using an analytical model and an extensive database of stabilities of individual proteins. Our results support the hypothesis that a cell's thermal sensitivities arise from the collective instability of its proteins. This model shows a denaturation catastrophe at temperatures of 49–55°C, roughly the thermal death point of mesophiles. Cells live on the edge of a proteostasis catastrophe. According to the model, it is not that the average protein is problematic; it is the tail of the distribution. About 650 of E. coli's 4300 proteins are less than 4 kcal mol−1 stable to denaturation. And upshifting by only 4° from 37° to 41°C is estimated to destabilize an average protein by nearly 20%. This model also treats effects of denaturants, osmolytes, and other physical stressors. In addition, it predicts the dependence of cellular growth rates on temperature. This approach may be useful for studying physical forces in biological evolution and the role of climate change on biology. PMID:21156142

In individual cells, transcription is a random process obeying single-molecule kinetics. Often, it occurs in a bursty, intermittent manner. The frequency and size of these bursts affect the magnitude of temporal fluctuations in mRNA and protein content within a cell, creating variation or “noise” in gene expression. It is still unclear to what degree transcriptional kinetics are specific to each gene and determined by its promoter sequence. Alternative scenarios have been proposed, where the kinetics of transcription are governed by cellular constraints and follow universal rules across the genome. Evidence from genome-wide noise studies and from systematic perturbations of promoter sequences suggest that both scenarios—namely gene-specific versus genome-wide regulation of transcription kinetics— may be present to different degrees in bacteria, yeast and animal cells. PMID:24311680

Early theoretical analysis predicted that microgravity effects on the isolated cell would be minuscule at the subcellular level; however, these speculations have not proven true in the real world. Astronauts experience a significant bone and muscle loss in as little as 2 weeks of spaceflight and changes are seen at the cellular level soon after exposure to microgravity. Changes in biological systems may be primarily due to the lack of gravity and the resulting loss of mechanical stress on tissues and cells. Recent ground and flight studies examining the effects of gravity or mechanical stress on cells demonstrate marked changes in gene expression when relatively small changes in mechanical forces or gravity fields were made. Several immediate early genes (IEG) like c-fos and c-myc are induced by mechanical stimulation within minutes. In contrast, several investigators report that the absence of mechanical forces during space flight result in decreased sera response element (SRE) activity and attenuation of expression of IEGs such as c-fos, c-jun and cox-2 mRNAs. Clearly, these early changes in gene expression may have long term consequences on mechanically sensitive cells. In our early studies on STS-56, we reported four major changes in the osteoblast; 1) prostaglandin synthesis in flight, 2) changes in cellular morphology, 3) altered actin cytoskeleton and 4) reduced osteoblast growth after four days exposure to microgravity. Initially, it was believed that changes in fibronectin (FN) RNA, FN protein synthesis or subsequent FN matrix formation might account for the changes in cytoskeleton and/ or reduction of growth. However our recent studies on Biorack (STS-76, STS-81 and STS-84), using ground and in-flight 1-G controls, demonstrated that fibronectin synthesis and matrix formation were normal in microgravity. In addition, in our most recent Biorack paper, our laboratory has documented that relative protein synthesis and mRNA synthesis are not changed after 24

Early theoretical analysis predicted that microgravity effects on the isolated cell would be minuscule at the subcellular level; however, these speculations have not proven true in the real world. Astronauts experience a significant bone and muscle loss in as little as 2 weeks of spaceflight and changes are seen at the cellular level soon after exposure to microgravity. Changes in biological systems may be primarily due to the lack of gravity and the resulting loss of mechanical stress on tissues and cells. Recent ground and flight studies examining the effects of gravity or mechanical stress on cells demonstrate marked changes in gene expression when relatively small changes in mechanical forces or gravity fields were made. Several immediate early genes (IEG) like c-fos and c-myc are induced by mechanical stimulation within minutes. In contrast, several investigators report that the absence of mechanical forces during space flight result in decreased sera response element (SRE) activity and attenuation of expression of IEGs such as c-fos, c-jun and cox-2 mRNAs. Clearly, these early changes in gene expression may have long term consequences on mechanically sensitive cells. In our early studies on STS-56, we reported four major changes in the osteoblast; 1) prostaglandin synthesis in flight, 2) changes in cellular morphology, 3) altered actin cytoskeleton and 4) reduced osteoblast growth after four days exposure to microgravity. Initially, it was believed that changes in fibronectin (FN) RNA, FN protein synthesis or subsequent FN matrix formation might account for the changes in cytoskeleton and/ or reduction of growth. However our recent studies on Biorack (STS-76, STS-81 and STS-84), using ground and in-flight 1-G controls, demonstrated that fibronectin synthesis and matrix formation were normal in microgravity. In addition, in our most recent Biorack paper, our laboratory has documented that relative protein synthesis and mRNA synthesis are not changed after 24

Carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) are glycoproteins produced in epithelial, endothelial, lymphoid, and myeloid cells. Carcinoembryonic antigen-related cellular adhesion molecules mediate cell-cell contact and host-pathogen interactions. The aims of this study were to map the distribution and examine the regulation of CEACAMs in human gingival sites. Quantitative real-time PCR performed on human gingival biopsies from periodontitis sites revealed mRNA coding for CEACAM1, -5, -6, and -7. Immunohistochemistry showed that CEACAMs were not found in oral gingival epithelium, except for CEACAM5 in periodontitis. Carcinoembryonic antigen-related cellular adhesion molecules 1, 5, and 6 were present in the oral sulcular epithelium of periodontitis but not in that of healthy gingiva. In junctional epithelium, all three molecules were present in healthy gingiva, but in periodontitis only CEACAM1 and -6 were detected. Staining for CEACAM1 and -6 was also seen in the inflammatory cell infiltrate in periodontitis. No staining for CEACAM7 was found. Proinflammatory mediators, including lipopolysaccharide (LPS), tumour necrosis factor-α (TNF-α)/interleukin-1β (IL-1β), and interferon-γ (IFN-γ), increased the expression of CEACAM1 and CEACAM6 mRNAs in cultured human oral keratinocytes. CEACAM1 and CEACAM6 mRNAs were also strongly up-regulated upon stimulation with lysophosphatidic acid. In conclusion, the distribution of different CEACAMs was related to specific sites in the gingiva. This might reflect different functional roles in this tissue.

The transcription factor gene Egr1 is necessary for female fertility; EGR1 protein is an established molecular regulator of adult female gonadotroph function where it mediates GNRH-stimulated transcription of the Lhb gene. Recent studies have also implicated pituitary EGR1 in the mediation of other physiological signals indicating an integrative function. However, the role of EGR1 in males is less well defined and this uncertainty is compounded by the absence of cellularexpression data in the male pituitary gland. The aim of this study, therefore, was to define the distribution of Egr1 gene expression in the adult male rat pituitary. To further this aim, we have evaluated cellular populations in a transgenic rat model (Egr1-d2EGFP), in which we demonstrate regulated green fluorescent protein (GFP) expression in EGR1+ pituitary cells. Cellular filling by GFP enabled morphological and molecular differentiation of different populations of gonadotrophs; Egr1 transcription and LHB were highly co-localised in a major population of large cells but only minimally co-localised in small GFP+ cells; the latter cells were shown to be largely (80%) composed of minority populations of GH+ somatotrophs (9% of total GH+) and PRL+ lactotrophs (3% of total PRL+). Egr1 transcription was not found in TSH+, ACTH+ or SOX2+ precursor cells and was only minimally co-localised in S-100β+ folliculostellate cells. Our demonstration that the Egr1 gene is actively and selectively transcribed in a major sub-population of male LHB+ cells indicates a largely conserved role in gonadotroph function and has provided a basis for further defining this role.

Western hemlock heartwood contains patches of tracheids with large amounts of cellular inclusions. Microscopic and chemical examination of the wood showed several types of deposits containing the lignans matairesinol, hydroxymatairesinol and conidendrin. The deposits, which were often relatively pure individual lignans, frequently assumed different physical forms and...

Copper is an essential but potentially harmful trace element required in many enzymatic processes involving redox chemistry. Cellular copper homeostasis in mammals is predominantly maintained by regulating copper transport through the copper import CTR proteins and the copper exporters ATP7A and ATP7B. Once copper is imported into the cell, several pathways involving a number of copper proteins are responsible for trafficking it specifically where it is required for cellular life, thus avoiding the release of harmful free copper ions. In this study we review recent progress made in understanding the molecular mechanisms of copper transport in cells by analyzing structural features of copper proteins, their mode of interaction, and their thermodynamic and kinetic parameters, thus contributing to systems biology of copper within the cell.

Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

Nanoparticles have been developed in broad biomedical research in terms of effective cellular interactions to treat and visualize diseased cells. Considering the charge and polar functional groups of proteins that are embedded in cellular membranes, charged nanoparticles have been strategically developed to enhance electrostatic cellular interactions. In this study, we show that cellular uptake efficiency, pathway, and spatial distribution of gold nanoparticles in a cell are significantly modulated based on the surface condition of gold nanoparticles and human cancer cells that were tuned by controlling the pH of the medium and by introducing an electron beam. Cellular uptake efficiency is increased when electrostatic attraction is induced between the cells and the gold nanoparticles. Cell surface modification changes the cellular uptake pathways of the gold nanoparticles and concentrates the gold nanoparticles at the membrane region. Surface modification of the gold nanoparticles also contributes to deep penetration and homogeneous spatial distributions in a cell.

Background The small RNAs (sRNA) are a regulatory class of RNA mainly represented by the 21 and 24-nucleotide size classes. The cellular sRNAs are processed by RNase III family enzyme dicer (Dicer like in plant) from a self-complementary hairpin loop or other type of RNA duplexes. The papaya genome has been sequenced, but its microRNAs and other regulatory RNAs are yet to be analyzed. Results We analyzed the genomic features of the papaya sRNA population from three sRNA deep sequencing libraries made from leaves, flowers, and leaves infected with Papaya Ringspot Virus (PRSV). We also used the deep sequencing data to annotate the micro RNA (miRNA) in papaya. We identified 60 miRNAs, 24 of which were conserved in other species, and 36 of which were novel miRNAs specific to papaya. In contrast to the Chargaff’s purine-pyrimidine equilibrium, cellular sRNA was significantly biased towards a purine rich population. Of the two purine bases, higher frequency of adenine was present in 23nt or longer sRNAs, while 22nt or shorter sRNAs were over represented by guanine bases. However, this bias was not observed in the annotated miRNAs in plants. The 21nt species were expressed from fewer loci but expressed at higher levels relative to the 24nt species. The highly expressed 21nt species were clustered in a few isolated locations of the genome. The PRSV infected leaves showed higher accumulation of 21 and 22nt sRNA compared to uninfected leaves. We observed higher accumulation of miRNA* of seven annotated miRNAs in virus-infected tissue, indicating the potential function of miRNA* under stressed conditions. Conclusions We have identified 60 miRNAs in papaya. Our study revealed the asymmetric purine-pyrimidine distribution in cellular sRNA population. The 21nt species of sRNAs have higher expression levels than 24nt sRNA. The miRNA* of some miRNAs shows higher accumulation in PRSV infected tissues, suggesting that these strands are not totally functionally redundant. The

The fragile X mental retardation protein (FMRP) plays an important role in normal brain development. Absence of FMRP, caused by transcriptional silencing of FMR1 gene, results in abnormal neuronal morphologies in a selected manner throughout the brain and leads to intellectual deficits and sensory dysfunction in the fragile X syndrome (FXS). Despite the paramount importance of FMRP for proper brain function, its overall expression pattern in the mammalian brain at the resolution of individual neuronal cell groups is not known. In this study, we used FMR1 knockout and their isogenic wild type mice to systematically map the distribution of FMRP expression in the entire mouse brain. Using immunocytochemistry and cellular quantification analyses, we identified a large number of prominent cell groups expressing high levels of FMRP at the subcortical levels, in particular sensory and motor neurons in the brainstem and thalamus. In contrast, many cell groups in the midbrain and hypothalamus exhibit low FMRP levels. More importantly, we describe differential patterns of FMRP distribution in both cortical and subcortical brain regions. Almost all major brain areas contain high and low levels of FMRP cell groups adjacent to each other or between layers of the same cortical areas. These differential patterns indicate that FMRP expression is specific to individual neuronal cell groups, instead of associated with brain regions as previously considered. Taken together, these findings support the notion that FMRP mechanisms of neuronal regulation are cell-type specific and strongly implicate the contribution of fundamental sensory and motor processing at subcortical levels to FXS pathology. PMID:27539535

Two synthetic hammerhead ribozymes, one unmodified and the other with 2"-modifications and four phosphorothioate groups, targeting a single GUA site in the luciferase mRNA, were compared for their inhibition of gene expression in cell cultureand their cellular uptake was also analysed. A HeLa X1/5 cell line stably expressing luciferase, under an inducible promoter, was treated with these ribozymes by liposome-mediated transfection to determine their activity.Luciferase expression in cells was inhibited to approximately 50% with little difference between the unmodified and the 2"-modified ribozyme. A similar degree of inhibition was observed with two catalytically inactive ribozymes, indicating that inhibition was mainly due to an antisense effect. A ribozyme carrying a cholesterol moiety, applied to the cells without carrier, showed no inhibition. Northern blotting indicated a similar amount of cellular uptake of all ribozymes. The unmodified ribozyme was essentially evenly distributed between cytoplasm and nucleus, whereas a higher proportion of the phosphorothioate-containing ribozyme was observed in the nucleus. Fluorescence microscopy, including confocal microscopy using 5"-fluorescein-labelled ribozymes, showed that the unmodified and 2"-modified ribozymes were present in the cytoplasm and in the nucleus to a similar extent, whereas the fluorescence of the phosphorothioate-containing ribozyme was much stronger in the nucleus. Both ribozymes inhibited luciferase expression to a comparable degree, suggesting that the ribozyme in the nucleus did not contribute significantly to the inhibition. Ribozymes with a cholesterol moiety were predominantly trapped in the cell membrane, explaining their inability to interfere with gene expression. PMID:10454613

The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation.

The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation. PMID:25984715

Stathmin is a ubiquitous cytosolic phosphoprotein, preferentially expressed in the nervous system, and previously described as a relay integrating diverse intracellular signaling pathways. Stathmin is the generic element of a mammalian protein family including SCG10, SCLIP, and RB3 with its splice variants RB3' and RB3". In contrast with stathmin, SCG10, SCLIP, and RB3/RB3'/RB3" are exclusively expressed in the nervous system, stathmin and SCG10 being mostly expressed during cell proliferation and differentiation, and SCLIP and RB3 rather in mature neural cells. To further understand their specific roles in the CNS, we compared the localization of the stathmin, SCG10, SCLIP, and RB3 transcripts in adult rat brain. Northern blot analysis as well as in situ hybridization experiments showed that all stathmin-related mRNAs are expressed in a wide range of adult rat brain areas. At a regional level, SCG10 and SCLIP appear generally distributed similarly except in a few areas. The pattern of expression of the RB3 transcript is very different from that of the three other members of the stathmin family. Furthermore, unlike SCG10 and SCLIP, which were detected only in neurons, but like stathmin, RB3 was detected in neurons and also in glial cells of the white matter. Altogether, our results suggest distinct roles for each member of the stathmin-related phosphoprotein family, in regard to their specific regional and cellular localization in the rat brain.

The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lower in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.

Heterogeneous and hygroscopic characteristics of plant-based food material make it complex in structure, and therefore water distribution in its different cellular environments is very complex. There are three different cellular environments, namely the intercellular environment, the intracellular environment, and the cell wall environment inside the food structure. According to the bonding strength, intracellular water is defined as loosely bound water, cell wall water is categorized as strongly bound water, and intercellular water is known as free water (FW). During food drying, optimization of the heat and mass transfer process is crucial for the energy efficiency of the process and the quality of the product. For optimizing heat and mass transfer during food processing, understanding these three types of waters (strongly bound, loosely bound, and free water) in plant-based food material is essential. However, there are few studies that investigate cellular level water distribution and transport. As there is no direct method for determining the cellular level water distributions, various indirect methods have been applied to investigate the cellular level water distribution, and there is, as yet, no consensus on the appropriate method for measuring cellular level water in plant-based food material. Therefore, the main aim of this paper is to present a comprehensive review on the available methods to investigate the cellular level water, the characteristics of water at different cellular levels and its transport mechanism during drying. The effect of bound water transport on quality of food product is also discussed. This review article presents a comparative study of different methods that can be applied to investigate cellular water such as nuclear magnetic resonance (NMR), bioelectric impedance analysis (BIA), differential scanning calorimetry (DSC), and dilatometry. The article closes with a discussion of current challenges to investigating cellular water

G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellularexpression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellularexpression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling. PMID:23139825

C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.

Monocarboxylate transporter 8 (MCT8) is a thyroid hormone (TH)-specific transporter. Mutations in the MCT8 gene are associated with Allan-Herndon-Dudley Syndrome (AHDS), consisting of severe psychomotor retardation and disturbed TH parameters. To study the functional consequences of different MCT8 mutations in detail, we combined functional analysis in different cell types with live-cell imaging of the cellulardistribution of seven mutations that we identified in patients with AHDS. We used two cell models to study the mutations in vitro: 1) transiently transfected COS1 and JEG3 cells, and 2) stably transfected Flp-in 293 cells expressing a MCT8-cyan fluorescent protein construct. All seven mutants were expressed at the protein level and showed a defect in T3 and T4 transport in uptake and metabolism studies. Three mutants (G282C, P537L, and G558D) had residual uptake activity in Flp-in 293 and COS1 cells, but not in JEG3 cells. Four mutants (G221R, P321L, D453V, P537L) were expressed at the plasma membrane. The mobility in the plasma membrane of P537L was similar to WT, but the mobility of P321L was altered. The other mutants studied (insV236, G282C, G558D) were predominantly localized in the endoplasmic reticulum. In essence, loss of function by MCT8 mutations can be divided in two groups: mutations that result in partial or complete loss of transport activity (G221R, P321L, D453V, P537L) and mutations that mainly disturb protein expression and trafficking (insV236, G282C, G558D). The cell type-dependent results suggest that MCT8 mutations in AHDS patients may have tissue-specific effects on TH transport probably caused by tissue-specific expression of yet unknown MCT8-interacting proteins.

Water channel aquaporin-4 (AQP4) is the most abundant water channel in the rodent brain and is mainly expressed in cerebral areas involved in central osmoreception and osmoregulation. The neurohypophysis is the release site of hypothalamic neurohormones vasopressin and oxytocin, which are involved in the regulation of the water balance. The authors investigated the cellular and subcellular distribution of AQP4 in the mouse neurohypophysis before and after chronic osmotic stimulation, using immunofluorescence microscopy and immunoperoxidase electron microscopy. They showed that AQP4 was abundant in the mouse hypophysis, mainly in the neural lobe. AQP4 was discontinuously distributed along pituicytes plasma membranes, in the dense neurosecretory granules and microvesicles of nerve endings and fibers, and along the luminal and abluminal membranes of fenestrated capillary endothelial cells. After chronic osmotic stimulation, AQP4 immunolabeling was enhanced. Taken together, these results suggest that AQP4 could be involved in the pituicyte sensor effect during osmoregulation, the modification and/or maturation mechanism of neurosecretory granules during neurohormone release, and the blood perfusion of the hypophysis.

Iron appears to be one of the main factors in the metal induced neurodegeneration. Quantitative information on cellular, sub-cellular and cell specific distributions of iron is therefore important to assess. The investigations reported here were carried out on a brain from an adult rat. Therefore, 6 μm thick embedded, unstained brain sections containing the midbrain (substantia nigra, SN) were analysed. Particle induced X-ray emission (PIXE) using a focussed proton beam (beam - diameter app. 1 μm) was performed to determine the quantitative iron content on a cellular and sub-cellular level. The integral analysis shows that the iron content in the SN pars reticulata is twice as high than in the SN pars compacta. The analysis of the iron content on the cellular level revealed no remarkable differences between glia cells and neurons. This is in contrast to other studies using staining techniques.

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain. PMID:23593173

The distribution of okadaic acid between the digestive and the secretory cells of the digestive gland of Mytilus galloprovincialis was studied for the main purpose of determining if they might be responsible for the two-compartment depuration kinetics found in previous studies. The two cell types did not accumulate okadaic acid to the same degree. However, the concentrations found in each cellular type were not consistent with those expected from the output of the two-compartment depuration model, suggesting that a mechanism other that the differential accumulation in cellular types is involved. Binding to some yet undetermined cellular components is suggested.

Dictyostelium discoideum encodes a single Rheb protein showing sequence similarity to human homologues of Rheb. The DdRheb protein shares 52 percent identity and 100 percent similarity with the human Rheb1 protein. Fluorescence of Rheb yellow fluorescent protein fusion was detected in the D. discoideum cytoplasm. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that rheb is expressed at all stages of development and in prestalk cells in the multicellular structures developed. When the expression of rheb as a fusion with lacZ was driven under its own promoter, the beta-galactosidase activity was seen in the prestalk cells. D. discoideum overexpressing Rheb shows an increase in the size of the cell. Treatment of the overexpressing Rheb cells with rapamycin confirms its involvement in the TOR signalling pathway.

It is widely recognized that radiopharmaceuticals are generally distributed nonuniformly in tissues. Such nonuniformities are observed over the entire range of spatial levels, ranging from organ to subcellular levels. The implications of nonuniform distributions of radioactivity for dosimetry, and ultimately for the biologic response of tissues containing radioactivity, have been investigated extensively. However, there is a paucity of experimental data on the distribution of cellular activity within a population of cells. In the present study, the distribution of activity per cell is experimentally determined and its implications for predicting biologic response are examined. Chinese hamster V79 cells were exposed to different concentrations of (210)Po-citrate. The radiolabeled cells were washed, seeded into culture dishes or glass slides, covered with photographic emulsion, and stored in an opaque container. Subsequently, the emulsion was developed, thereby resulting in observable alpha-particle tracks that were scored. The distribution of activity per cell was found to be well described by a log normal distribution function. Theoretic modeling of cell survival as a function of mean activity per cell showed that survival curves differed substantially when the activity per cell was log normally distributed versus when it was assumed conventionally that every cell in the population contained the mean activity. The present study provides experimental evidence of log normal cellular uptake of radioactivity. Theoretic calculations show that a log normal distribution of cellular activity can have a substantial impact on modeling the biologic response of cell populations.

Regional hypoxia caused by accelerated cell proliferation and overgrowth is an important characteristic of neoplasm. Hypoxia can cause a series of changes in gene transcription and protein expression, thereby not only inducing tumor cell resistance to radiotherapy and chemotherapy but also promoting tumor invasion and metastasis. This study aimed to investigate the relationship between HIF-1α expression and cellular apoptosis, angiogenesis and clinical prognosis in rectal carcinoma. In 113 rectal carcinoma cases, cellular apoptosis was analyzed by the in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, whereas the levels of HIF-1α expression, VEGF expression, microvessel density (MVD) and lymphatic vessel density(LVD) were examined by immunohistochemical staining. HIF-1 expression was detected in 67 of 113 rectal carcinoma cases (59.3 %). A positive correlation was found among HIF-1α expression, cellular apoptosis and angiogenesis. The 5-year survival rate in the HIF-1α-negative group was significantly higher than that in the HIF-1α-positive group (81.34 % versus 50 %, P expression, VEGF expression and cellular apoptosis index were independent risk factors for clinical prognosis in rectal carcinoma. Aberrant HIF-1α expression correlates with apoptosis inhibition, angiogenesis and poor prognosis in rectal carcinoma.

The recent description of a prion disease (PD) case in a free-ranging bottlenose dolphin (Tursiops truncatus) prompted us to carry out an extensive search for the disease-associated isoform (PrPSc) of the cellular prion protein (PrPC) in the brain and in a range of lymphoid tissues from 23 striped dolphins (Stenella coeruleoalba), 5 bottlenose dolphins and 2 Risso s dolphins (Grampus griseus) found stranded between 2007 and 2012 along the Italian coastline. Three striped dolphins and one bottlenose dolphin showed microscopic lesions of encephalitis, with no evidence of spongiform brain lesions being detected in any of the 30 free-ranging cetaceans investigated herein. Nevertheless, we could still observe a prominent PrPC immunoreactivity in the brain as well as in lymphoid tissues from these dolphins. Although immunohistochemical and Western blot investigations yielded negative results for PrPSc deposition in all tissues from the dolphins under study, the reported occurrence of a spontaneous PD case in a wild dolphin is an intriguing issue and a matter of concern for both prion biology and intra/inter-species transmissibility, as well as for cetacean conservation medicine.

Patients with esophageal squamous cell carcinoma (ESCC) have a poor prognosis. However, the related mechanisms are unclear, thus we investigated the expression of HO-1 in ESCC tissue and explored possible mechanisms of tumor progression. Expression of HO-1 was examined by immunohistochemistry in 143 ESCC tumors. The correlation of HO-1 with clinicopathological characteristics was also examined. Two human ESCC cell lines, TE-13 and Eca109 were studied. Silencing of cell line HO-1 by specific small interfering RNA (siRNA) was evaluated using real-time quantitative PCR. Cell line viability, apoptosis and intracellular levels of reactive oxygen species (ROS) after transfection were determined using MTT and flow cytometry, respectively. HO-1, Bax, Bcl-2 and A-caspase-3/-9 expression was evaluated using western blot analyses. We found that HO-1 was expressed in 58 of 143 ESCC tumors, mainly in the cytoplasm. There was a significant association between HO-1 expression and tumor grade (P<0.001). Knockdown of HO-1 expression in cell lines was associated with significantly decreased cellular proliferation (P<0.05) and a higher rate of apoptosis (P<0.001) 48 h after treatment. Treatment of the cell lines with the ROS inhibitor N-acetylcysteine abrogated this effect. Knockdown of HO-1 was associated with increased A-caspase-3 and -9 expression, but no change in Bax or Bcl-2 expression or Bax/Bcl-2 ratio was observed. Thus, the present study identified that ESCC tumors frequently expressed HO-1. Knockdown of HO-1 promoted apoptosis through activation of a ROS-mediated caspase apoptosis pathway.

Nicotinic and serotoninergic 5HT3 receptors share important sequence identities except for their cytoplasmic loop. Both ends of this loop display conserved 3D helical structures with distinct primary sequences. We decided to check whether these two helices named F and G play a role in the sub-cellulardistribution of different nicotinic receptors. We systematically exchanged each helix with the equivalent sequence of neuronal nicotinic and alpha4, beta2 and alpha7 subunits in the functional chimeric alpha7-5HT3 receptor used as a model system. The new chimeras were expressed in vitro in polarized epithelial cells from pig kidney. We quantified synthesis and export of the receptors to the cell surface by measuring alpha-bungarotoxin binding sites. Immunogold labelling was used, at the electron microscope level, to determine the amount of each chimera present at either domain, apical and/or basolateral, of these cells. We noticed that in epithelial cells the majority of alpha-bungarotoxin binding sites remained sequestered in the cytoplasm as already observed in neurons in vivo. The majority of the pentamers present at the cell surface were located at the apical domain. Our results suggest that helix F and G differently regulate assembly and export to the cell surface of alpha-bungarotoxin binding receptors.

Infrared microspectroscopy (IMS) and chemical imaging is ideal for measuring nutrient distribution in single algal cells on a cellular and subcellular level. The study of small algal cells, or cells within a colony requires enhanced spatial resolution IMS. Synchrotron infrared microspectroscopy wit...

Objective: To investigate whether differentiation or cellular confluence is responsible for CXCL11 expression patterns in re-epithelialization. Approach: In vitro model systems of re-epithelialization using the HaCaT keratinocyte cell line were utilized in monitoring expression of differentiation markers, including desmoplakin and various cytokeratins while evaluating for an association with chemokine CXCL11 expression. Results: CXCL11 expression was elevated in sparse culture with peak expression near the time of confluence. This somewhat followed the accumulation of desmoplakin in detergent-insoluble pool of proteins. However, in postconfluent, despite continued accumulation of desmoplakin within cells, CXCL11 expression decreased to baseline levels. This biphasic pattern was also seen in low calcium culture, an environment that inhibits keratinocyte differentiation and accumulation of desmosomal proteins. Highest CXCL11-expressing areas best correlated with newly confluent areas within culture expressing basal keratin 14, but also activated keratin 6. Innovation: Achievement of a threshold cellular density induces cell signaling cascade through CXCR3 that, in addition to other undiscovered pathways, can progress cutaneous wounds from the proliferative into the remodeling phases of cutaneous wound healing. Conclusion: These results suggest that the achievement of confluence with increased cellular density by migrating keratinocytes at the wound edge triggers expression of CXCL11. Since CXCR3 stimulation in endothelial cells results in apoptosis and causes neovascular pruning, whereas stimulation of CXCR3 in fibroblasts results decreased motility and cellular contraction, we speculate that CXCL11 expression by epidermal cells upon achieving cellular confluence could be the source of CXCR3 stimulation in the dermis ushering a transition from proliferative to remodeling phases of wound healing. PMID:28078185

ETS factors have been shown to be dysregulated in breast cancer. ETS factors control the expression of genes involved in many biological processes, such as cellular proliferation, differentiation, and apoptosis. FLI1 is an ETS protein aberrantly expressed in retrovirus-induced hematological tumors, but limited attention has been directed towards elucidating the role of FLI1 in epithelial-derived cancers. Using data mining, we show that loss of FLI1 expression is associated with shorter survival and more aggressive phenotypes of breast cancer. Gain and loss of function cellular studies indicate the inhibitory effect of FLI1 expression on cellular growth, migration, and invasion. Using Fli1 mutant mice and both a transgenic murine breast cancer model and an orthotopic injection of syngeneic tumor cells indicates that reduced Fli1 contributes to accelerated tumor growth. Global expression analysis and RNA-Seq data from an invasive human breast cancer cell line with over expression of either FLI1 and another ETS gene, PDEF, shows changes in several cellular pathways associated with cancer, such as the cytokine-cytokine receptor interaction and PI3K-Akt signaling pathways. This study demonstrates a novel role for FLI1 in epithelial cells. In addition, these results reveal that FLI1 down-regulation in breast cancer may promote tumor progression. PMID:25379017

Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements. PMID:25779048

The putative tumor suppressor gene spinophilin has been involved in cancer progression in several types of cancer. In this study, we explored the prognostic value of spinophilin expression in 162 colon adenocarcinoma patients. In addition, we generated stably expressing spinophilin-directed shRNA CRC cell lines and studied the influence of spinophilin expression on cellular phenotypes and molecular interactions. We independently confirmed that low spinophilin expression levels are associated with poor prognosis in CRC patients (p = 0.038). A reduction of spinophilin levels in p53 wild-type HCT116 and p53-mutated Caco-2 cells led to increased cellular growth rates and anchorage-independent growth (p<0.05). At molecular level, reduced spinophilin levels increased the expression of the transcription factor E2F-1. In addition, we observed an increased formation of tumor spheres, increased number of CD133 positive cells and an increased resistance to 5-flourouracil (p<0.05). Finally, treatment with the de-methylating agent 5-aza-dC increased spinophilin expression in CRC cells (p<0.05), corroborated by a correlation of spinophilin expression and extent of methylated CpG sites in the gene promoter region (p<0.001). In conclusion, gain of aggressive biological properties of CRC cells including cellular growth, cancer stem cell features and 5-flourouracil resistance partly explains the role of spinophilin in CRC. PMID:25261368

Recent advances have substantially increased the number of genes that are statistically associated with complex genetic disorders of the CNS such as autism and schizophrenia. It is now clear that there will likely be hundreds of distinct loci contributing to these disorders, underscoring a remarkable genetic heterogeneity. It is unclear whether this genetic heterogeneity indicates an equal heterogeneity of cellular mechanisms for these diseases. The commonality of symptoms across patients suggests there could be a functional convergence downstream of these loci upon a limited number of cell types or circuits that mediate the affected behaviors. One possible mechanism for this convergence would be the selective expression of at least a subset of these genes in the cell types that comprise these circuits. Using profiling data from mice and humans, we have developed and validated an approach, cell type-specific expression analysis, for identifying candidate cell populations likely to be disrupted across sets of patients with distinct genetic lesions. Using human genetics data and postmortem gene expression data, our approach can correctly identify the cell types for disorders of known cellular etiology, including narcolepsy and retinopathies. Applying this approach to autism, a disease where the cellular mechanism is unclear, indicates there may be multiple cellular routes to this disorder. Our approach may be useful for identifying common cellular mechanisms arising from distinct genetic lesions. PMID:24453331

Recently, the distribution of radioactivity among a population of cells labeled with 210Po was shown to be well described by a lognormal (LN) distribution function (J Nucl Med. 2006;47:1049-1058) with the aid of autoradiography. To ascertain the influence of Poisson statistics on the interpretation of the autoradiographic data, the present work reports on a detailed statistical analysis of these earlier data. The measured distributions of alpha-particle tracks per cell were subjected to statistical tests with Poisson, LN, and Poisson-lognormal (P-LN) models. The LN distribution function best describes the distribution of radioactivity among cell populations exposed to 0.52 and 3.8 kBq/mL of 210Po-citrate. When cells were exposed to 67 kBq/mL, the P-LN distribution function gave a better fit; however, the underlying activity distribution remained lognormal. The present analysis generally provides further support for the use of LN distributions to describe the cellular uptake of radioactivity. Care should be exercised when analyzing autoradiographic data on activity distributions to ensure that Poisson processes do not distort the underlying LN distribution.

Recently, the distribution of radioactivity among a population of cells labeled with 210Po was shown to be well described by a log normal distribution function (J Nucl Med 47, 6 (2006) 1049-1058) with the aid of an autoradiographic approach. To ascertain the influence of Poisson statistics on the interpretation of the autoradiographic data, the present work reports on a detailed statistical analyses of these data. Methods The measured distributions of alpha particle tracks per cell were subjected to statistical tests with Poisson (P), log normal (LN), and Poisson – log normal (P – LN) models. Results The LN distribution function best describes the distribution of radioactivity among cell populations exposed to 0.52 and 3.8 kBq/mL 210Po-citrate. When cells were exposed to 67 kBq/mL, the P – LN distribution function gave a better fit, however, the underlying activity distribution remained log normal. Conclusions The present analysis generally provides further support for the use of LN distributions to describe the cellular uptake of radioactivity. Care should be exercised when analyzing autoradiographic data on activity distributions to ensure that Poisson processes do not distort the underlying LN distribution. PMID:16741316

Recently, the distribution of radioactivity among a population of cells labeled with 210Po was shown to be well described by a log-normal (LN) distribution function (J Nucl Med. 2006;47:1049–1058) with the aid of autoradiography. To ascertain the influence of Poisson statistics on the interpretation of the autoradiographic data, the present work reports on a detailed statistical analysis of these earlier data. Methods The measured distributions of α-particle tracks per cell were subjected to statistical tests with Poisson, LN, and Poisson-lognormal (P-LN) models. Results The LN distribution function best describes the distribution of radioactivity among cell populations exposed to 0.52 and 3.8 kBq/mL of 210Po-citrate. When cells were exposed to 67 kBq/mL, the P-LN distribution function gave a better fit; however, the underlying activity distribution remained log-normal. Conclusion The present analysis generally provides further support for the use of LN distributions to describe the cellular uptake of radioactivity. Care should be exercised when analyzing autoradiographic data on activity distributions to ensure that Poisson processes do not distort the underlying LN distribution. PMID:18483086

The human immunodeficiency virus type I (HIV-1) has developed several strategies to condition the host environment to promote viral replication and spread. Viral proteins have evolved to perform multiple functions, aiding in the replication of the viral genome and modulating the cellular response to the infection. Tat is a small, versatile, viral protein that controls transcription of the HIV genome, regulates cellular gene expression and generates a permissive environment for viral replication by altering the immune response and facilitating viral spread to multiple tissues. Studies carried out utilizing biochemical, cellular, and genomic approaches show that the expression and activity of hundreds of genes and multiple molecular networks are modulated by Tat via multiple mechanisms.

Self-assembly of small molecules, as a more common phenomenon than one previously thought, can be either beneficial or detrimental to cells. Despite its profound biological implications, how the self-assembly of small molecules behave in a cellular environment is largely unknown and barely explored. This work studies four fluorescent molecules that consist of the same peptidic backbone (e.g., Phe-Phe-Lys) and enzyme trigger (e.g., a phosphotyrosine residue), but bear different fluorophores on the side chain of the lysine residue of the peptidic motif. These molecules, however, exhibit a different ability of self-assembly before and after enzymatic transformation (e.g., dephosphorylation). Fluorescent imaging reveals that self-assembly directly affects the distribution of these small molecules in a cellular environment. Moreover, cell viability tests suggest that the states and the locations of the molecular assemblies in the cellular environment control the phenotypes of the cells. For example, the molecular nanofibers of one of the small molecules apparently stabilize actin filaments and alleviate the insult of an F-actin toxin (e.g., latrunculin A). Combining fluorescent imaging and enzyme-instructed self-assembly of small peptidic molecules, this work demonstrates self-assembly as a key factor for dictating the spatial distribution of small molecules in a cellular environment. In addition, it illustrates a useful approach, based on enzyme-instructed self-assembly of small molecules, to modulate spatiotemporal profiles of small molecules in a cellular environment, which allows the use of the emergent properties of small molecules to control the fate of cells.

Somatostatin (SST), a neuropeptide expressed in dendritic-targeting gamma-aminobutyric acid (GABA) neurons, is decreased across corticolimbic areas in major depressive disorder (MDD). SST-positive GABA neurons form heterogeneous subgroups with different laminar distributions and electrophysiological properties, so knowing the anatomical and cellular localization of reduced SST may provide insight into the nature of the pathology in MDD. In cohorts of MDD subjects with known reduction of SST in postmortem sgACC gray matter, we used in situ hybridization to quantify the laminar and cellular patterns of altered SST mRNA expression. SST mRNA levels were lower across all cortical layers in the MDD subjects. Expression levels per cell were also lower, but the density of labeled neurons did not differ between subject groups. Consistent with the previous tissue level analysis, differences were more robust in females. In summary, we report MDD-related reduction in SST expression per cell across cortical layers in sgACC, suggesting a general vulnerability of SST neurons independent of specific cell type. PMID:25315685

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Recently, an extensive layer of intra-cellular signals was discovered that was previously undetected by genetic radar. It is now known that this layer consists primarily of a class of short noncoding RNA species that are referred to as microRNAs (miRNAs). MiRNAs regulate protein synthesis at the post-transcriptional level, and studies have shown that they are involved in many fundamental cellular processes. In this study, we hypothesized that miRNAs may be involved in cellular stress response mechanisms, and that cells exposed to thermal stress may exhibit a signature miRNA expression profile indicative of their functional involvement in such mechanisms. To test our hypothesis, human dermal fibroblasts were exposed to an established hyperthermic protocol, and the ensuing miRNA expression levels were evaluated 4 hr post-exposure using microRNA microarray gene chips. The microarray data shows that 123 miRNAs were differentially expressed in cells exposed to thermal stress. We collectively refer to these miRNAs as thermalregulated microRNAs (TRMs). Since miRNA research is in its infancy, it is interesting to note that only 27 of the 123 TRMs are currently annotated in the Sanger miRNA registry. Prior to publication, we plan to submit the remaining novel 96 miRNA gene sequences for proper naming. Computational and thermodynamic modeling algorithms were employed to identify putative mRNA targets for the TRMs, and these studies predict that TRMs regulate the mRNA expression of various proteins that are involved in the cellular stress response. Future empirical studies will be conducted to validate these theoretical predictions, and to further examine the specific role that TRMs play in the cellular stress response.

Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellulardistributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

Background Brain injury results in an increase in the activity of the reactive oxygen species generating NADPH oxidase (NOX) enzymes. Preliminary studies have shown that NOX2, NOX3, and NOX4 are the most prominently expressed NOX isotypes in the brain. However, the cellular and temporal expression profile of these isotypes in the injured and non-injured brain is currently unclear. Methods Double immunofluorescence for NOX isotypes and brain cell types was performed at acute (24 hours), sub-acute (7 days), and chronic (28 days) time points after controlled cortical impact-induced brain injury or sham-injury in rats. Results NOX2, NOX3, and NOX4 isotypes were found to be expressed in neurons, astrocytes, and microglia, and this expression was dependent on both cellular source and post-injury time. NOX4 was found in all cell types assessed, while NOX3 was positively identified in neurons only, and NOX2 was identified in microglia and neurons. NOX2 was the most responsive to injury, increasing primarily in microglia in response to injury. Quantitation of this isotype showed a significant increase in NOX2 expression at 24 hours, with reduced expression at 7 days and 28 days post-injury, although expression remained above sham levels at later time points. Cellular confirmation using purified primary or cell line culture demonstrated similar patterns in microglia, astrocytes, and neurons. Further, inhibition of NOX, and more specifically NOX2, reduced pro-inflammatory activity in microglia, demonstrating that NOX is not only up-regulated after stimulation, but may also play a significant role in post-injury neuroinflammation. Conclusions This study illustrates the expression profiles of NOX isotypes in the brain after injury, and demonstrates that NOX2, and to a lesser extent, NOX4, may be responsible for the majority of oxidative stress observed acutely after traumatic brain injury. These data may provide insight into the design of future therapeutic approaches. PMID

Expression profiles of genes involved in cell wall metabolism and water transport were compared with changes in grape (Vitis vinifera L.) berry growth, basic chemical composition, and the shape, size, and wall thickness of cells within tissues of the berry pericarp. Expression of cell wall-modifying and aquaporin genes in berry pericarp tissues generally followed a bimodal expression profile with high levels of expression coinciding with the two periods of rapid berry growth, stages I and III, and low levels of expression corresponding to the slow-growth period, stage II. Cellular expansion was observed throughout all tissues during stage I, and only mesocarp cellular expansion was observed during stage III. Expansion of only exocarp cells was evident during transition between stages II and III. Cell wall-modifying and aquaporin gene expression profiles followed similar trends in exocarp and mesocarp tissues throughout berry development, with the exception of the up-regulation of pectin methylesterase, pectate lyase, two aquaporin genes (AQ1 and AQ2), and two expansin genes (EXP3 and EXPL) during stage II, which was delayed in the exocarp tissue compared with mesocarp tissue. Exocarp endo-(1-->3)-beta-glucanase and expansin-like gene expression was concurrent with increases in epidermal and hypodermal cell wall thickness. These results indicate a potential role of the grape berry skin in modulating grape berry growth.

The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

Cellular processes are "noisy". In each cell, concentrations of molecules are subject to random fluctuations due to the small numbers of these molecules and to environmental perturbations. While noise varies with time, it is often measured at steady state, for example by flow cytometry. When interrogating aspects of a cellular network by such steady-state measurements of network components, a key need is to develop efficient methods to simulate and compute these distributions. We describe innovations in stochastic modeling coupled with approaches to this computational challenge: first, an approach to modeling intrinsic noise via solution of the chemical master equation, and second, a convolution technique to account for contributions of extrinsic noise. We show how these techniques can be combined in a streamlined procedure for evaluation of different sources of variability in a biochemical network. Evaluation and illustrations are given in analysis of two well-characterized synthetic gene circuits, as well as a signaling network underlying the mammalian cell cycle entry.

Arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder, liver and lung. Inorganic arsenic, existing in highly toxic trivalent and significantly less toxic pentavalent forms, is methylated to mono- and di-methylated species mainly in the liver. Due to the low toxicity of pentavalent methylated species, methylation has been regarded as a detoxification process for many years; however, recent findings of a high toxicity of trivalent methylated species have indicated the contrary. In order to elucidate the role of speciation and methylation for the toxicity and carcinogenicity of arsenic, systematic studies were conducted comparing cellular uptake, subcellular distribution as well as toxic and genotoxic effects of organic and inorganic pentavalent and trivalent arsenic species in both non-methylating (urothelial cells and fibroblasts) and methylating cells (hepatocytes). The membrane permeability was found to be dependent upon both the arsenic species and the cell type. Uptake rates of trivalent methylated species were highest and exceeded those of their pentavalent counterparts by several orders of magnitude. Non-methylating cells (urothelial cells and fibroblasts) seem to accumulate higher amounts of arsenic within the cell than the methylating hepatocytes. Cellular uptake and extrusion seem to be faster in hepatocytes than in urothelial cells. The correlation of uptake with toxicity indicates a significant role of membrane permeability towards toxicity. Furthermore, cytotoxic effects are more distinct in hepatocytes. Differential centrifugation studies revealed that elevated concentrations of arsenic are present in the ribosomal fraction of urothelial cells and in nucleic and mitochondrial fractions of hepatic cells. Further studies are needed to define the implications of the observed enrichment of arsenic in specific cellular organelles for its carcinogenic activity. This review summarizes our recent research on cellular uptake

We intend to know the protein expression of genetic alterations that take place in the early stages in the field cancerization of oral cavity in our means as well as to study the cellular proliferation by means of Ki-67 and the protein product expression of p53 to value if the alterations in the protein products expression of these markers happen in a sequential pathway through the different stages in the field cancerization of oral cavity. A study was made by immunohistochemistry on 53 patients that presented lesions of oral leukoplaquia, assisted by the ENT service at University Hospital of Salamanca, from 1.990 up to 2000. 11 samples of normal epithelium, 15 mild to moderate dysplasias, 15 in situ carcinomas and 12 microinvasive carcinomas are included in the study. we find an increased cellular proliferation and p53 over-expression as we advance in the grade of severity histopathologic of these lesions. The most early alterations are a significant increase of cell proliferation in mild and moderate dysplasias and an increased p53 over-expression. Oral leukoplaquia is a precancerous stage that constitutes a cancerisable lesion due to the genetic alterations that mediate in the evolution of lesion. Routine Immunohistochemical and molecular study of these lesions allow us to know the protein expression of genetic alterations that can help in the early diagnosis and treatment of this pathology, having special relevance the study of Ki-67 in early stages and p53 in advanced lesions.

The spread of an infectious disease is determined by biological and social factors. Models based on cellular automata are adequate to describe such natural systems consisting of a massive collection of simple interacting objects. They characterize the time evolution of the global system as the emergent behaviour resulting from the interaction of the objects, whose behaviour is defined through a set of simple rules that encode the individual behaviour and the transmission dynamic. An epidemic is characterized trough an individual-based-model built upon cellular automata. In the proposed model, each individual of the population is represented by a cell of the automata. This way of modeling an epidemic situation allows to individually define the characteristic of each individual, establish different scenarios and implement control strategies. A cellular automata model to study the time evolution of a heterogeneous populations through the various stages of disease was proposed, allowing the inclusion of individual heterogeneity, geographical characteristics and social factors that determine the dynamic of the desease. Different assumptions made to built the classical model were evaluated, leading to following results: i) for low contact rate (like in quarantine process or low density population areas) the number of infective individuals is lower than other areas where the contact rate is higher, and ii) for different initial spacial distributions of infected individuals different epidemic dynamics are obtained due to its influence on the transition rate and the reproductive ratio of disease. The contact rate and spatial distributions have a central role in the spread of a disease. For low density populations the spread is very low and the number of infected individuals is lower than in highly populated areas. The spacial distribution of the population and the disease focus as well as the geographical characteristic of the area play a central role in the dynamics of the

Background The spread of an infectious disease is determined by biological and social factors. Models based on cellular automata are adequate to describe such natural systems consisting of a massive collection of simple interacting objects. They characterize the time evolution of the global system as the emergent behaviour resulting from the interaction of the objects, whose behaviour is defined through a set of simple rules that encode the individual behaviour and the transmission dynamic. Methods An epidemic is characterized trough an individual–based–model built upon cellular automata. In the proposed model, each individual of the population is represented by a cell of the automata. This way of modeling an epidemic situation allows to individually define the characteristic of each individual, establish different scenarios and implement control strategies. Results A cellular automata model to study the time evolution of a heterogeneous populations through the various stages of disease was proposed, allowing the inclusion of individual heterogeneity, geographical characteristics and social factors that determine the dynamic of the desease. Different assumptions made to built the classical model were evaluated, leading to following results: i) for low contact rate (like in quarantine process or low density population areas) the number of infective individuals is lower than other areas where the contact rate is higher, and ii) for different initial spacial distributions of infected individuals different epidemic dynamics are obtained due to its influence on the transition rate and the reproductive ratio of disease. Conclusions The contact rate and spatial distributions have a central role in the spread of a disease. For low density populations the spread is very low and the number of infected individuals is lower than in highly populated areas. The spacial distribution of the population and the disease focus as well as the geographical characteristic of the area

Recent studies have provided evidence that a local renin-angiotensin system (RAS) exists in the retina and plays an important role in retinal neurovascular function. We have recently shown that increased expression of ACE2 and angiotensin (1-7) [Ang (1-7)], two components of the protective axis of the RAS, in the retina via adeno-associated virus (AAV)-mediated gene delivery, conferred protection against diabetes-induced retinopathy. We hypothesized that the protective molecular and cellular mechanisms of Ang (1-7) are mediated by its receptor, Mas, and the expression level and cellular localization dictate the response to Ang (1-7) and activation of subsequent protective signaling pathways. We tested this hypothesis by examining the expression and cellular localization of the Mas receptor in adult and developing mouse retinas. The cellular localization of the Mas receptor protein was determined with immunofluorescence of the eyes of adult and postnatal day 1 (P1), P5, P7, P15, and P21 mice using the Mas receptor-specific antibody, and mRNA was detected with in situ hybridization of paraffin-embedded sections. Western blotting and real-time reverse-transcription (RT)-PCR analysis were performed to determine the relative levels of the Mas protein and mRNA in adult and developing retinas, as well as in cultured retinal Müller glial and RPE cells. In the adult eye, the Mas receptor protein was abundantly present in retinal ganglion cells (RGCs) and photoreceptor cells; a lower level of expression was observed in endothelial cells, Müller glial cells, and other neurons in the inner nuclear layer of the retina. In the developing retina, Mas receptor mRNA and protein expression was detected in the inner retina at P1, and the expression levels increased with age to reach the adult level and pattern by P15. In the adult mouse retina, Mas receptor mRNA was expressed at a much higher level when compared to angiotensin II (Ang II) type I (AT1R) and type II (AT2R) receptor m

Animals comprise dynamic three-dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy based approaches to establish permanent, quantitative datasets—atlases—that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization and quantitative analysis. PMID:24123936

Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

We examined the effects of total body exposure of BCF1 mice to {gamma}-rays (300 cGy) in modulating expression of cellular oncogenes in both gut and liver tissues. We selected specific cellular oncogenes (c-fos, c-myc, c-src, and c-H-ras), based on their normal expression in liver and gut tissues from untreated mice. As early as 5 min. following whole body exposure of BCF1 mice to {gamma}-rays we detected induction of mRNA specific for c-src and c-H-ras in both liver and gut tissues. c-fos RNA was slightly decreased in accumulation in gut but was unaffected in liver tissue from irradiated mice relative to untreated controls. c-myc mRNA accumulation was unaffected in all tissues examined. These experiments document that modulation of cellular oncogene expression can occur as an early event in tissues following irradiation and suggest that this modulation may play a role in radiation-induced carcinogenesis.

X-chromosome linked inhibitor of apoptosis, XIAP, is cellular caspase inhibitor and a key regulator of apoptosis. We and others have previously shown that XIAP expression is regulated primarily at the level of protein synthesis; the 5′ untranslated region (UTR) of XIAP mRNA contains an Internal Ribosome Entry Site (IRES) that supports cap-independent expression of XIAP protein during conditions of pathophysiological stress, such as serum deprivation or gamma irradiation. Here, we show that XIAP is encoded by two distinct mRNAs that differ in their 5′ UTRs. We further show that the dominant, shorter, 5′ UTR promotes a basal level of XIAP expression under normal growth conditions. In contrast, the less abundant longer 5′ UTR contains an IRES and supports cap-independent translation during stress. Our data suggest that the combination of alternate regulatory regions and distinct translational initiation modes is critical in maintaining XIAP levels in response to cellular stress and may represent a general mechanism of cellular adaptation. PMID:20385593

The c-myc oncogene has been implicated in the development of many different cancers, yet the mechanism by which the c-myc protein alters cellular growth control has proven elusive. The authors used a cDNA hybridization difference assay to isolate two genes, mr1 and mr2, that were constitutively expressed (i.e., deregulated) in rodent fibroblast cell lines immortalized by transfection of a viral promoter-linked c-myc gene. Both cDNAs were serum inducible in quiescent G/sub o/ fibroblasts, suggesting that they are functionally related to cellular proliferative processes. Although there were significant differences in cytoplasmic mRNA levels between myc-immortalized and control cells, the rates of transcription and mRNA turnover of both genes were similar, suggesting that c-myc regulates mr1 and mr2 expression by some nuclear posttranscriptional mechanism. Their results provide evidence that c-myc can rapidly modulate cellular gene expression and suggest that c-myc may function in gene regulation at the level of RNA export, splicing, or nuclear RNA turnover.

The cellulardistribution and temporal expression of transcripts from transforming growth factor-beta 1 (TGF-beta 1) and procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) genes were studied in carbon tetrachloride (CCl4)-induced rat liver fibrosis by using in situ hybridization technique. During the fibrotic process, TGF-beta 1 and procollagen genes were similarly and predominantly expressed in Desmin-positive perisinusoidal cells (e.g., fat-storing cells and myofibroblasts) and fibroblasts and their expression continued to be higher than those observed in control rats. These transcripts were also observed in inflammatory cells mainly granulocytes and macrophage-like cells at the early stages of liver fibrosis. The production of extracellular matrix along small blood vessels and fibrous septa coincided with the expression of these genes. Expression of TGF-beta 1 and procollagen genes were not detected in hepatocytes throughout the experiment. No significant differences in cellulardistribution or time course of gene expression among procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) were observed. Desmin-positive perisinusoidal cells and fibroblasts appeared to play the principal role in synthesis of collagens in CCl4-induced hepatic fibrosis. The simultaneous expression of TGF-beta 1 and procollagen genes in mesenchymal cells, including Desmin-positive perisinusoidal cells, during hepatic fibrosis suggests the possibility that TGF-beta 1 may have an important role in the production of fibrosis. Images PMID:1693377

An ensemble of stochastic genetic relaxation oscillators via phase-attractive or repulsive cell-to-cell communication are investigated. In the phase-attractive coupling case, it is found that cellular communication can enhance self-induced stochastic resonance as well as collective rhythms, and that different intensities of noise resulting from the fluctuation of intrinsic chemical reactions or the extrinsic environment can induce stochastic limit cycles with different amplitudes for a large cell density. In contrast, in the phase-repulsive coupling case, the distribution of phase differences among the stochastic oscillators can display such characteristic as unimodality, bimodality or polymodality, depending on both noise intensity and cell number, but the modality of phase difference distribution almost keeps invariant for an arbitrary noise intensity as the cell number is beyond a threshold.

The cellular prion protein (PrP(c)) is a highly conserved glycoprotein with a still enigmatic physiological function. It is mainly expressed in the central nervous system but accumulating data suggest that PrP(c) is also found in a broad spectrum of non-neuronal tissue. Here we investigated the cell-type-related PrP(c) expression in the bovine mammary gland by using immunohistochemistry (IHC), ELISA, Western blot, and real-time RT-PCR. Specific immunostaining of serial sections revealed that PrP(c) is selectively localized in mammary gland epithelial cells. Particularly strong expression was found at the basolateral surface of those cells showing active secretion. Results obtained by RT-PCR and ELISA complemented IHC findings. No correlation was found between the level of PrP(c) expression and other parameters such as age of the animals under study or stage of lactation.

The purpose of this study was to investigate whether intracellular distribution of Na+, K+-ATPase α3 subunit, a receptor for cardiac glycosides including oleandrin, is differentially altered in cancer versus normal cells and whether this altered distribution can be therapeutically targeted to inhibit cancer cell survival. The cellulardistribution of Na+, K+-ATPase α3 isoform was investigated in paired normal and cancerous mucosa biopsy samples from patients with lung and colorectal cancers by immunohistochemical staining. The effects of oleandrin on α3 subunit intracellular distribution, cell death, proliferation, and EKR phosphorylation were examined in differentiated and undifferentiated human colon cancer CaCO-2 cells. While Na+, K+-ATPase α3 isoform was predominantly located near the cytoplasmic membrane in normal human colon and lung epithelia, the expression of this subunit in their paired cancer epithelia was shifted to a peri-nuclear position in both a qualitative and quantitative manner. Similarly, distribution of α3 isoform was also shifted from a cytoplasmic membrane location in differentiated human colon cancer CaCO-2 cells to a peri-nuclear position in undifferentiated CaCO-2 cells. Intriguingly, oleandrin exerted threefold stronger anti-proliferative activity in undifferentiated CaCO-2 cells (IC50, 8.25 nM) than in differentiated CaCO-2 cells (IC50, >25 nM). Oleandrin (10 to 20 nM) caused an autophagic cell death and altered ERK phosphorylation in undifferentiated but not in differentiated CaCO-2 cells. These data demonstrate that the intracellular location of Na+, K+-ATPase α3 isoform is altered in human cancer versus normal cells. These changes in α3 cellular location and abundance may indicate a potential target of opportunity for cancer therapy. PMID:23073998

The structure and the expression of 11 cellular oncogenes (protooncogenes) were analyzed in primary cells from 20 acute lymphocytic (ALL) and 31 acute myelogenous (AML) leukemia patients. Neoplastic cells, obtained prior to initiation of therapy, were purified and classified, on the basis of both surface antigen pattern and morphology, into pre-B, B, and T ALL and M1-M5 AML. RNA was extracted and analyzed for expression of cellular oncogenes coding for nuclear proteins (c-myc, c-myb, c-fos), the beta-chain of platelet-derived growth factor (c-sis), growth factor receptors or related proteins (c-src, c-abl, c-fes, c-erbB), or putative intermediate transducers of mitogenic signals (c-Ha-ras, c-Ki-ras, c-N-ras). Quantitative analysis of total RNA was carried out by dot blot hybridization to specific cDNA or genomic probes. Number and size of transcripts were evaluated by blot hybridization of electrophoretically fractionated poly(A)+ RNA. Expression of c-myc and c-myb was detected in all leukemic cells at variable levels and was characterized by well-defined patterns within ALL subtypes. Conversely, significant levels of c-fos transcripts were detected only in myelomonocytic (M4) and monocytic (M5) leukemias. Among the "src-family," c-fes was expressed more in AML than ALL, and c-abl was expressed at variable but not elevated levels in all leukemia types. c-Ha-ras was uniformly expressed at low levels, as in non-neoplastic cells. c-Ki-ras transcription was detected only in T ALL; N-ras expression was barely demonstrable. The structure of these protooncogenes was not grossly modified, as evaluated by Southern analysis, except for c-myc rearrangement in B ALL. These studies indicate that cellular oncogene expression in specific subtypes of leukemic cells may relate to either the proliferative activity (c-myc, c-myb) or the differentiation state (c-fos) of the cells, or possibly to expression of receptors for putative hemopoiesis-related growth factors (c-fes, c

Fibronectin (Fn) regulates cell migration, proliferation, and extracellular matrix formation during embryogenesis, angiogenesis, and wound healing. Fn also promotes mesangial cell migration and proliferation in vitro and contributes to extracellular matrix formation and tissue remodeling during glomerular disease. In this study, we examined, by immunohistochemistry and in situ hybridization, the temporal glomerular localization and cellular sources of Fn in Habu snake venom (HSV)-induced proliferative glomerulonephritis. Early HSV-induced glomerular lesions consisted of microaneurysms devoid of resident glomerular cells and filled with platelets, leukocytes, and erythrocytes. Over the course of the disease, mesangial cells migrated into the lesions, proliferated, and formed a confluent cellular mass. Fn was present in lesions beginning at 8 hours, with highest intensity at 72 hours and diminishing at 2 weeks after HSV. Staining for Fn at 8 and 24 hours after HSV was attributed to platelets and macrophages. In situ hybridization and phenotypic identification of cell types within lesions revealed macrophages as the predominant source of cellular Fn mRNA at these times. At 48 hours after HSV, Fn mRNA was expressed in proliferating mesangial cells in addition to macrophages. Most cells in lesions at 72 hours after HSV were mesangial, at a time when expression of Fn mRNA peaked. Cellularexpression for Fn mRNA and translated protein declined at 2 weeks after HSV. These studies support the hypothesis that Fn, derived from platelets and macrophages, provides a provisional matrix involved with mesangial cell migration into glomerular lesions. Fn produced by mesangial cells might contribute to the formation of a stable extracellular matrix. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8080041

The objective of this study was to determine the molecular mechanisms responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients. Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double-strand break (DSB) repair in cells. Quantitative PCR (Q-PCR) established the expression levels of key DNA DSB repair genes. Imaging flow cytometry using annexin V-FITC was used to compare artemis expression and apoptosis in cells. Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. The Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene, which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Overexpression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis. We conclude that elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity.

Background: The objective of this study was to determine the molecular mechanisms responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients. Methods: Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double-strand break (DSB) repair in cells. Quantitative PCR (Q-PCR) established the expression levels of key DNA DSB repair genes. Imaging flow cytometry using annexin V-FITC was used to compare artemis expression and apoptosis in cells. Results: Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. The Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene, which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Overexpression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis. Conclusion: We conclude that elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity. PMID:23093295

Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

To ensure efficient energy supply to the high demanding brain, nutrients are transported into brain cells via specific glucose (GLUT) and monocarboxylate transporters (MCT). Mitochondrial dysfunction and altered glucose metabolism are thought to play an important role in the progression of neurodegenerative diseases, including multiple sclerosis (MS). Here, we investigated the cellular localization of key GLUT and MCT proteins in human brain tissue of non-neurological controls and MS patients. We show that in control brain tissue GLUT and MCT proteins were abundantly expressed in a variety of central nervous system cells, particularly in microglia and endothelial cells. In active MS lesions, GLUTs and MCTs were highly expressed in infiltrating leukocytes and reactive astrocytes. Astrocytes manifest increased MCT1 staining and maintain GLUT expression in inactive lesions, whereas demyelinated axons exhibit significantly reduced GLUT3 and MCT2 immunoreactivity in inactive lesions. Finally, we demonstrated that the co-transcription factor peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), an important protein involved in energy metabolism, is highly expressed in reactive astrocytes in active MS lesions. Overexpression of PGC-1α in astrocyte-like cells resulted in increased production of several GLUT and MCT proteins. In conclusion, we provide for the first time a comprehensive overview of key nutrient transporters in white matter brain samples. Moreover, our data demonstrate an altered expression of these nutrient transporters in MS brain tissue, including a marked reduction of axonal GLUT3 and MCT2 expression in chronic lesions, which may impede efficient nutrient supply to the hypoxic demyelinated axons thereby contributing to the ongoing neurodegeneration in MS.

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. The virus adopts a strategy based on the lack of viral expression in vivo; only very rare BLV-infected B lymphocytes express viral information. When the cells are isolated from animals in persistent lymphocytosis and cultivated ex vivo, a tremendous increase in viral expression occurs. To gain insight into this mechanism, we employed a general approach using chemicals that interfere specifically with cellular pathways involved in signal transduction from the cell membrane to the nucleus. Our data demonstrate that BLV expression is not correlated with the activity of protein kinase A (PKA) and is even inhibited by cyclic AMP (cAMP). The cAMP/PKA pathway is thus apparently not involved in ex vivo viral expression. In contrast, PKC appears to play a key role in this process. Phorbol myristate acetate can directly activate viral expression in B cells (in the absence of T cells). Furthermore, calphostin C, a highly specific inhibitor of PKC, partly decreases ex vivo BLV expression. Our data further demonstrate that calmodulin and calcineurin, a calmodulin-dependent phosphatase, play a key role in the induction of viral expression. The involvement of this calmodulin-dependent pathway could explain the induction of expression that cannot be assigned to PKC. Furthermore, it appears that the activation of viral expression requires a calmodulin but not a PKA-dependent pathway. These data highlight major differences between transient transfection and ex vivo experiments. Finally, despite their homologies, BLV and human T-cell leukemia virus appear to use different signal transduction pathways to induce viral expression. PMID:8642639

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A gene's “expression profile” denotes the number of transcripts present relative to all other transcripts. The overall rate of transcript production is determined by transcription and RNA processing rates. While the speed of elongating RNA polymerase II has been characterized for many different genes and organisms, gene-architectural features – primarily the number and length of exons and introns – have recently emerged as important regulatory players. Several new studies indicate that rapidly cycling cells constrain gene-architecture toward short genes with a few introns, allowing efficient expression during short cell cycles. In contrast, longer genes with long introns exhibit delayed expression, which can serve as timing mechanisms for patterning processes. These findings indicate that cell cycle constraints drive the evolution of gene-architecture and shape the transcriptome of a given cell type. Furthermore, a tendency for short genes to be evolutionarily young hints at links between cellular constraints and the evolution of animal ontogeny. PMID:25400101

In Drosophila, the Trithorax-group (trxG) and Polycomb-group (PcG) proteins interact with chromosomal elements, termed Cellular Memory Modules (CMMs). By modifying chromatin, this ensures a stable heritable maintenance of the transcriptional state of developmental regulators, like the homeotic genes, that is defined embryonically. We asked whether such CMMs could also control expression of genes involved in patterning imaginal discs during larval development. Our results demonstrate that expression of the hedgehog gene, once activated, is maintained by a CMM. In addition, our experiments indicate that the switching of such CMMs to an active state during larval stages, in contrast to embryonic stages, may require specific trans-activators. Our results suggest that the patterning of cells in particular developmental fields in the imaginal discs does not only rely on external cues from morphogens, but also depends on the previous history of the cells, as the control by CMMs ensures a preformatted gene expression pattern.

The heat stress (HS)-induced increase in occludin protein expression has been postulated to be a protective response against HS-induced disruption of the intestinal epithelial tight junction barrier. The aim of this study was to elucidate the cellular and molecular processes that mediate the HS-induced up-regulation of occludin expression in Caco-2 cells. Exposure to HS (39°C or 41°C) resulted in increased expression of occludin protein; this was preceded by an increase in occludin mRNA transcription and promoter activity. HS-induced activation of heat shock factor-1 (HSF-1) resulted in cytoplasmic-to-nuclear translocation of HSF-1 and binding to its binding motif in the occludin promoter region. HSF-1 activation was associated with an increase in occludin promoter activity, mRNA transcription, and protein expression; which were abolished by the HSF-1 inhibitor quercetin. Targeted HSF-1 knock-down by siRNA transfection inhibited the HSF-1-induced increase in occulin expression and junctional localization of occulin protein. Site-directed mutagenesis of the HSF-1 binding motif in the occludin promoter region inhibited HS-induced binding of HSF-1 to the occludin promoter region and subsequent promoter activity. In conclusion, our data show for the first time that the HS-induced increase in occludin protein expression is mediated by HSF-1 activation and subsequent binding of HSF-1 to the occludin promoter, which initiates a series of molecular and cellular events culminating in increased junctional localization of occludin protein. PMID:18276783

Cellular senescence of biliary epithelial cells with p16INK4a and p21WAF1/Cip expression in damaged small bile ducts may be critical for progressive bile duct loss in primary biliary cirrhosis. We investigated the involvement of bmi1, a polycomb group gene repressing p16INK4a expression, in the pathogenesis of biliary cellular senescence. Bmi1 expression was examined immunohistochemically in livers taken from the patients with primary biliary cirrhosis (n = 18) and other diseased (n = 19) and normal livers (n = 16). We examined the effect of oxidative stress and a short interference RNA (siRNA) targeting bmi1 on cellular senescence in cultured mouse biliary epithelial cells. Bmi1 was widely expressed in the nuclei of biliary epithelial cells in the control livers. In contrast, bmi1 expression was significantly decreased in damaged small bile ducts in 43% of livers with primary biliary cirrhosis of stage 1/2, coordinating with the increased p16INK4a expression. In cultured biliary epithelial cells, oxidative stress by H2O2 treatment significantly decreased bmi1 expression, followed by increased P16INK4a expression. A knockdown of bmi1 induced increased p16INK4a expression, decreased cell proliferation, and increased cellular senescence. In conclusion, the decreased bmi1 expression caused by oxidative stress may be involved in the pathogenesis of cellular senescence of biliary epithelial cells in primary biliary cirrhosis. PMID:16936260

Bleomycin-induced pulmonary fibrosis is associated with increased lung transforming growth factor-beta (TGF-beta) gene expression, but cellular localization of the source of this expression has not been unequivocally established. In this study, lung fibrosis was induced in rats by endotracheal bleomycin injection on day 0 and, on selected days afterwards, lungs were harvested for in situ hybridization, immunohistochemical and histochemical analyses for TGF-beta 1 mRNA and protein expression, and cell identification. The results show that control lungs express essentially no detectable TGF-beta 1 mRNA or protein in the parenchyma. Before day 3 after bleomycin treatment, scattered bronchiolar epithelial cells, mononuclear cells, and eosinophils expressed elevated levels of TGF-beta 1. Between days 3 and 14, there was a major increase in the number of eosinophils, myofibroblasts, and fibroblasts strongly expressing TGF-beta 1 mRNA and protein. TGF-beta 1-producing cells were predominantly localized within areas of injury and active fibrosis. After day 14, the intensity and number of TGF-beta 1-expressing cells significantly declined and were predominantly found in fibroblasts in fibrotic areas. The expression of TGF-beta 1 protein was generally coincident with that for mRNA with the exception of bronchiolar epithelial cells in which strong protein expression was unaccompanied by a commensurate increase in mRNA. The study demonstrates that myofibroblasts, fibroblasts, and eosinophils represent the major sources of increased lung TGF-beta 1 expression in this model of pulmonary fibrosis. Images Figure 2 Figure 3 Figure 4 PMID:7543734

The interplay between cellular adhesion and proliferation is complex; however, integrins, particularly the α5β1 subset, play a pivotal role in orchestrating critical cellular signals that culminate in cellular adhesion and growth. Retinoids modify the expression of a variety of adhesive/proliferative signaling proteins including α5β1 integrins; however, the role of specific retinoic acid receptors involved in these processes has not been elucidated. In this study, the effect of all-trans-retinoic acid receptor (RAR) agonists on K562 cellular adhesion, proliferation, and α5β1 integrin cell surface expression was investigated. RARγ agonist exposure increased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin and FN-120 in a time- and concentration dependent manner, while RARα or RARβ agonist treatment had no effect on cellular adhesion. Due to the novel RARγ- dependent cellular adhesion response exhibited by K562 cells, we examined α5 and β1 integrin subunit expression when K562 cells were exposed to retinoid agonists or vehicle for 24, 48, 72 or 96 hours. Our data demonstrates no differences in K562 cell surface expression of the α5 integrin subunit when cells were exposed to RARα, RARβ, or RARγ agonists for all time points tested. In contrast, RARγ agonist exposure resulted in an increase in cell surface β1 integrin subunit expression within 48 hours that was sustained at 72 and 96 hours. Finally, we demonstrate that while exposure to RARα or RARβ agonists have no effect on K562 cellular proliferation, the RARγ agonist significantly dampens K562 cellular proliferation levels in a time- and concentration- dependent manner. Our study is the first to report that treatment with a RARγ specific agonist augments cellular adhesion to α5β1 integrin substrates, increases cell surface levels of the β1 integrin subunit, and dampens cellular proliferation in a time and concentration dependent manner in a human

Zea mays (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellulardistribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellulardistribution of ZmO2 may have important functional consequences for ZmO2 activity.

Zea mays (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellulardistribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellulardistribution of ZmO2 may have important functional consequences for ZmO2 activity. PMID:22937104

The pathways governing signal transduction in the mesocortical and nigrostriatal dopamine systems of the brain are of central importance in a variety of drug actions and neurological diseases. We have analyzed the regional, cellular, and subcellular distribution of the closely related D1 and D5 subtypes of dopamine receptors in the cerebral cortex and selected subcortical structures of rhesus monkey using subtype specific antibodies. The distribution of D1 and D5 receptors was highly differentiated in subcortical structures. In the neostriatum, both D1 and to a lesser extent D5 antibodies labeled medium spiny neurons, while only D5 antibodies labeled the large aspiny neurons typical of cholinergic interneurons. In the caudate nucleus, D1 labeling was concentrated in the spines and shafts of projection neurons, whereas D5 antibodies predominantly labeled the shafts, and less commonly, the spines of these cells. The D1 receptor was abundantly expressed in the neuropil of the substantia nigra pars reticulata while the D5 antibodies labeled only a few scattered cell bodies in this structure. Conversely, D5 antibodies labeled cholinergic neurons in the basal forebrain more intensely than D1 antibodies. Within the cerebral cortex and hippocampus, D1 and D5 antibody labeling was prominent in pyramidal cells. Double-label experiments revealed that the two receptors were frequently coexpressed in neurons of both structures. Ultrastructurally, D1 receptors were especially prominent in dendritic spines whereas dendritic shafts were more prominently labeled by the D5 receptor. The anatomical segregation of the D1 and D5 receptors at the subcellular level in cerebral cortex and at the cellular level in subcortical areas suggest that these closely related receptors may be preferentially associated with different circuit elements and may play distinct regulatory roles in synaptic transmission.

The intrinsic stochasticity of gene expression can lead to a large variability of protein levels across a population of cells. Variability (or noise) in protein distributions can be modulated by cellular mechanisms of gene regulation; in particular, there is considerable interest in understanding the role of post-transcriptional regulation. To address this issue, we propose and analyze a stochastic model for post-transcriptional regulation of gene expression. The analytical solution of the model provides insight into the effects of different mechanisms of post-transcriptional regulation on the noise in protein distributions. The results obtained also demonstrate how different sources of intrinsic noise in gene expression can be discriminated based on observations of regulated protein distributions.

The intrinsic stochasticity of gene expression can lead to a large variability of protein levels across a population of cells. Variability (or noise) in protein distributions can be modulated by cellular mechanisms of gene regulation; in particular, there is considerable interest in understanding the role of post-transcriptional regulation. To address this issue, we propose and analyze a stochastic model for post-transcriptional regulation of gene expression. The analytical solution of the model provides insight into the effects of different mechanisms of post-transcriptional regulation on the noise in protein distributions. The results obtained also demonstrate how different sources of intrinsic noise in gene expression can be discriminated based on observations of regulated protein distributions.

Cellular processes are “noisy”. In each cell, concentrations of molecules are subject to random fluctuations due to the small numbers of these molecules and to environmental perturbations. While noise varies with time, it is often measured at steady state, for example by flow cytometry. When interrogating aspects of a cellular network by such steady-state measurements of network components, a key need is to develop efficient methods to simulate and compute these distributions. We describe innovations in stochastic modeling coupled with approaches to this computational challenge: first, an approach to modeling intrinsic noise via solution of the chemical master equation, and second, a convolution technique to account for contributions of extrinsic noise. We show how these techniques can be combined in a streamlined procedure for evaluation of different sources of variability in a biochemical network. Evaluation and illustrations are given in analysis of two well-characterized synthetic gene circuits, as well as a signaling network underlying the mammalian cell cycle entry. PMID:22022252

The amount of cellular proteins is a crucial parameter that is known to vary between cells as a function of the replicative passages, and can be important during physiological aging. The process of protein degradation is known to be performed by a series of enzymatic reactions, ranging from an initial step of protein ubiquitination to their final fragmentation by the proteasome. In this paper we propose a stochastic dynamical model of nuclear proteins concentration resulting from a balance between a constant production of proteins and their degradation by a cooperative enzymatic reaction. The predictions of this model are compared with experimental data obtained by fluorescence measurements of the amount of nuclear proteins in murine tail fibroblast (MTF) undergoing cellular senescence. Our model provides a three-parameter stationary distribution that is in good agreement with the experimental data even during the transition to the senescent state, where the nuclear protein concentration changes abruptly. The estimation of three parameters (cooperativity, saturation threshold, and maximal velocity of the reaction), and their evolution during replicative passages shows that only the maximal velocity varies significantly. Based on our modeling we speculate the reduction of functionality of the protein degradation mechanism as a possible competitive inhibition of the proteasome. PMID:26115222

Potassium (K(+)) is the most important cationic nutrient for all living organisms. Its cellular levels are significant (typically around 100mM) and are highly regulated. In plants K(+) affects multiple aspects such as growth, tolerance to biotic and abiotic stress and movement of plant organs. These processes occur at the cell, organ and whole plant level and not surprisingly, plants have evolved sophisticated mechanisms for the uptake, efflux and distribution of K(+) both within cells and between organs. Great progress has been made in the last decades regarding the molecular mechanisms of K(+) uptake and efflux, particularly at the cellular level. For long distance K(+) transport our knowledge is less complete but the principles behind the overall processes are largely understood. In this chapter we will discuss how both long distance transport between different organs and intracellular transport between organelles works in general and in particular for K(+). Where possible, we will provide examples of specific genes and proteins that are responsible for these phenomena.

We report the uptake of MSNs into the roots and their movement to the aerial parts of four plant species and their quantification using fluorescence, TEM and proton-induced x - ray emission (micro - PIXE) elemental analysis. Monodispersed mesoporous silica nanoparticles (MSNs) of optimal size and configuration were synthesized for uptake by plant organs, tissues and cells. These monodispersed nanoparticles have a size of 20 nm with interconnected pores with an approximate diameter of 2.58 nm. There were no negative effects of MSNs on seed germination or when transported to different organs of the four plant species tested in this study. Most importantly, for the first time, a combination of confocal laser scanning microscopy, transmission electron microscopy and proton-induced X-ray emission (micro-PIXE) elemental analysis allowed the location and quantification MSNs in tissues and in cellular and sub-cellular locations. Our results show that MSNs penetrated into the roots via symplastic and apoplastic pathways and then via the conducting tissues of the xylem to the aerial parts of the plants including the stems and leaves. The translocation and widescale distribution of MSNs in plants will enable them to be used as a new delivery means for the transport of different sized biomolecules into plants.

The amount of cellular proteins is a crucial parameter that is known to vary between cells as a function of the replicative passages, and can be important during physiological aging. The process of protein degradation is known to be performed by a series of enzymatic reactions, ranging from an initial step of protein ubiquitination to their final fragmentation by the proteasome. In this paper we propose a stochastic dynamical model of nuclear proteins concentration resulting from a balance between a constant production of proteins and their degradation by a cooperative enzymatic reaction. The predictions of this model are compared with experimental data obtained by fluorescence measurements of the amount of nuclear proteins in murine tail fibroblast (MTF) undergoing cellular senescence. Our model provides a three-parameter stationary distribution that is in good agreement with the experimental data even during the transition to the senescent state, where the nuclear protein concentration changes abruptly. The estimation of three parameters (cooperativity, saturation threshold, and maximal velocity of the reaction), and their evolution during replicative passages shows that only the maximal velocity varies significantly. Based on our modeling we speculate the reduction of functionality of the protein degradation mechanism as a possible competitive inhibition of the proteasome.

We used a single-molecule localization technique called direct stochastic optical reconstruction microscopy (dSTORM) to quantify both colocalization and spatial distribution on a cellular level for two conceptually different N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates. Microscopy images were acquired of entire cells with resolutions as high as 25 nm revealing the nanoscale distribution of the fluorescently labeled therapeutic components. Drug-free macromolecular therapeutics consisting of two self-assembling nanoconjugates showed slight increase in nanoclusters on the cell surface with time. Additionally, dSTORM provided high resolution images of the nanoscale organization of the self-assembling conjugates at the interface between two cells. A conjugate designed for treating ovarian cancer showed that the model drug (Cy3) and polymer bound to Cy5 were colocalized at an early time point before the model drug was enzymatically cleaved from the polymer. Using spatial descriptive statistics it was found that the drug was randomly distributed after 24 h while the polymer bound dye remained in clusters. Four different fluorescent dyes were used and two different therapeutic systems were tested to demonstrate the versatility and possible general applicability of dSTORM for use in studying drug delivery systems. PMID:26855050

BACKGROUND: Myocarditis and dilated cardiomyopathy (DCM) are common causes of morbidity and mortality in children and adults. Recently, the human coxsackievirus and adenovirus receptor (CAR), a common receptor for coxsackieviruses and adenoviruses, was discovered and its increased expression has been reported in patients with DCM and myocarditis. OBJECTIVE: To measure the expression of CAR in myocardial tissues of patients with DCM and its cellular localization in DCM cases. METHODS: Formalin-fixed myocardial tissues collected during autopsy from 26 cases of DCM, and 20 cases each of noncardiac disease and cardiac disease other than DCM were included as the test group, and control groups A and B, respectively. Expression of CAR was studied using immunohistochemical staining of myocardial tissue with a CAR-specific rabbit polyclonal antibody. CAR messenger RNA was semiquantified by reverse transcription polymerase chain reaction followed by agarose gel analysis and measurement of band intensity. RESULTS: CAR positivity in DCM cases was found to be 96% (25 of 26) compared with 30% in control group A and 40% in control group B. CAR was found to be expressed in myocytes, endothelial and interstitial cells; however, positivity in myocytes was significantly higher than in other cells in all groups. The site of CAR expression was predominantly the sarcolemma along with cytoplasm in cardiomyocytes. CONCLUSIONS: The present study highlighted the increased expression of CAR in DCM cases, with localization in myocytes and endothelial cells. PMID:23592932

Recent studies on fractionation of carbon isotopes in biological systems are reviewed. It follows that direct experimental proofs have been obtained that 1) basic fractionation of carbon isotopes in the cell is related to isotope effect in pyruvate decarboxylation; 2) fractionation of carbon isotopes in the above reaction in vivo proceeds with exhausting substrate pool. The latter provides natural relationship between metabolites isotope distribution and sequence of their synthesis in the cell cycle, or with the temporal organization of cellular metabolism. The non-steady and periodic pattern of pyruvate decarboxylation due to the exhausting substrate pool well agrees with the existing notions on reciprocal oscillations in the cell glycolytic chain. Experimental data are presented corroborating indirectly the existence of oscillations in bacterial cells. Earlier proposed model of the mechanism of carbon isotope fractionation based on the above principles can be used for analysing changes in isotopic characteristics of the organisms and interpreting their relations with metabolic processes.

Nanoparticle-assisted drug delivery has been emerging as an active research area. Achieving high drug loading is only one facet of drug delivery issues; it is also important to investigate the effect of surface charge distribution on self-assembly of nanoparticles on cellular membranes. By considering the electrostatic distribution of patterned nanoparticles, we used dissipative particle dynamics simulations to investigate the self-assembly of pattern charged nanoparticles with five different surface charged patterns. It is found that both surface charged pattern and nanoparticle size significantly affect the self-assembly of nanoparticles on cellular membranes. Results indicate that 1/2 pattern charged small nanoparticles can self-assemble into dendritic structures, while those with a 1/4 pattern self-assemble into clusters. As the nanoparticle size increases, 1/2 pattern charged medium nanoparticles can self-assemble into linear structures, while those with a 1/4 pattern self-assemble into clusters. For very large nanoparticles, both 1/2 pattern and 1/4 pattern charged nanoparticles self-assemble into flaky structures with different connections. By considering the effects of surface charged pattern and nanoparticle size on self-assembly, we found that nanoparticle self-assembly requires a minimum effective charged area. When the local charged area of nanoparticles is less than the threshold, surface charge cannot induce nanoparticle self-assembly; that is, the surface charged pattern of a nanoparticle would determine effectively the self-assembly structure. It is expected that this work will provide guidance for nanoparticle-assisted drug delivery.

The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.

In rheumatoid arthritis (RA), a key event is infiltration of inflammatory immune cells into the synovial lining, possibly aggravated by dysregulation of cellular adhesion molecules. Therefore, single nucleotide polymorphisms of 14 genes involved in cellular adhesion processes (CAST, ITGA4, ITGB1, ITGB2, PECAM1, PTEN, PTPN11, PTPRC, PXN, SELE, SELP, SRC, TYK2, and VCAM1) were analyzed for association with RA. Association analysis was performed consecutively in three European RA family sample groups (Nfamilies = 407). Additionally, we investigated differential allelic expression, a possible functional consequence of genetic variants. SELP (selectin P, CD62P) SNP-allele rs6136-T was associated with risk for RA in two RA family sample groups as well as in global analysis of all three groups (ptotal = 0.003). This allele was also expressed preferentially (p<10−6) with a two- fold average increase in regulated samples. Differential expression is supported by data from Genevar MuTHER (p1 = 0.004; p2 = 0.0177). Evidence for influence of rs6136 on transcription factor binding was also found in silico and in public datasets reporting in vitro data. In summary, we found SELP rs6136-T to be associated with RA and with increased expression of SELP mRNA. SELP is located on the surface of endothelial cells and crucial for recruitment, adhesion, and migration of inflammatory cells into the joint. Genetically determined increased SELP expression levels might thus be a novel additional risk factor for RA. PMID:25147926

Although a physiological function of the cellular prion protein (PrP(c)) is still not fully clarified, a PrP(c)-mediated neuroprotection against hypoxic/ischemic insult is intriguing. After ischemic stroke prion protein knockout mice (Prnp(0/0)) display significantly greater lesions as compared to wild-type (WT) mice. Earlier reports suggested an interaction between the glycolytic enzyme lactate dehydrogenase (LDH) and PrP(c). Since hypoxic environment enhances LDH expression levels and compels neurons to rely on lactate as an additional oxidative substrate for energy metabolism, we examined possible differences in LDH protein expression in WT and Prnp(0/0) knockout models under normoxic/hypoxic conditions in vitro and in vivo, as well as in a HEK293 cell line. While no differences are observed under normoxic conditions, LDH expression is markedly increased after 60-min and 90-min of hypoxia in WT vs. Prnp(0/0) primary cortical neurons with concurrent less hypoxia-induced damage in the former group. Likewise, cerebral ischemia significantly increases LDH levels in WT vs. Prnp(0/0) mice with accompanying smaller lesions in the WT group. HEK293 cells overexpressing PrP(c) show significantly higher LDH expression/activity following 90-min of hypoxia as compared to control cells. Moreover, a cytoplasmic co-localization of LDH and PrP(c) was recorded under both normoxic and hypoxic conditions. Interestingly, an expression of monocarboxylate transporter 1, responsible for cellular lactate uptake, increases with PrP(c)-overexpression under normoxic conditions. Our data suggest LDH as a direct PrP(c) interactor with possible physiological relevance under low oxygen conditions.

The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of γ-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca2+-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. PMID:15912504

Purpose Recent studies have provided evidence that a local renin-angiotensin system (RAS) exists in the retina and plays an important role in retinal neurovascular function. We have recently shown that increased expression of ACE2 and angiotensin (1-7) [Ang (1-7)], two components of the protective axis of the RAS, in the retina via adeno-associated virus (AAV)-mediated gene delivery, conferred protection against diabetes-induced retinopathy. We hypothesized that the protective molecular and cellular mechanisms of Ang (1-7) are mediated by its receptor, Mas, and the expression level and cellular localization dictate the response to Ang (1-7) and activation of subsequent protective signaling pathways. We tested this hypothesis by examining the expression and cellular localization of the Mas receptor in adult and developing mouse retinas. Methods The cellular localization of the Mas receptor protein was determined with immunofluorescence of the eyes of adult and postnatal day 1 (P1), P5, P7, P15, and P21 mice using the Mas receptor-specific antibody, and mRNA was detected with in situ hybridization of paraffin-embedded sections. Western blotting and real-time reverse-transcription (RT)–PCR analysis were performed to determine the relative levels of the Mas protein and mRNA in adult and developing retinas, as well as in cultured retinal Müller glial and RPE cells. Results In the adult eye, the Mas receptor protein was abundantly present in retinal ganglion cells (RGCs) and photoreceptor cells; a lower level of expression was observed in endothelial cells, Müller glial cells, and other neurons in the inner nuclear layer of the retina. In the developing retina, Mas receptor mRNA and protein expression was detected in the inner retina at P1, and the expression levels increased with age to reach the adult level and pattern by P15. In the adult mouse retina, Mas receptor mRNA was expressed at a much higher level when compared to angiotensin II (Ang II) type I (AT1R) and

X-ray irradiation influences metastatic properties of tumor cells and, moreover, metastasis and cellular motility can be modified by members of the Eph receptor/ephrin family of receptor tyrosine kinases. We hypothesized that irradiation-induced changes in cellular properties relevant for metastasis in melanoma cells could be mediated by Eph receptor/ephrin signaling. In this pilot study, we analyzed one pre-metastatic (Mel-Juso) and three metastatic human melanoma (Mel-Juso-L3, A375, and A2058) cells lines and predominantly found anti-metastatic effects of X-ray irradiation with impaired cell growth, clonal growth and motility. Additionally, we observed an irradiation-induced increase in adhesion paralleled by a decrease in migration in Mel-Juso and Mel-Juso-L3 cells and, in part, also in A375 cells. We further demonstrate a decrease of EphA2 both in expression and activity at 7 d after irradiation paralleled by an upregulation of EphA3. Analyzing downstream signaling after irradiation, we detected decreased Src kinase phosphorylation, but unchanged focal adhesion kinase (FAK) phosphorylation, indicating, in part, irradiation-induced downregulation of signaling via the EphA2-Src-FAK axis in melanoma cells. However, to which extent this finding contributes to the modification of metastasis-relevant cellular properties remains to be elucidated. PMID:22568947

Motivation: Genome-scale metabolic models have been used extensively to investigate alterations in cellular metabolism. The accuracy of these models to represent cellular metabolism in specific conditions has been improved by constraining the model with omics data sources. However, few practical methods for integrating metabolomics data with other omics data sources into genome-scale models of metabolism have been reported. Results: GIMMME (Gene Inactivation Moderated by Metabolism, Metabolomics, and Expression) is an algorithm that enables the development of condition-specific models based on an objective function, transcriptomics, and intracellular metabolomics data. GIMMME establishes metabolite utilization requirements with metabolomics data, uses model-paired transcriptomics data to find experimentally supported solutions, and also provides calculations of the turnover (production / consumption) flux of metabolites. GIMMME was employed to investigate the effects of integrating additional omics datasets to create increasingly constrained solution spaces of Salmonella Typhimurium metabolism during growth in both rich and virulence media. This integration proved to be informative and resulted in a requirement of additional active reactions (12 in each case) or metabolites (26 or 29, respectively). The addition of constraints from transcriptomics also impacted the allowed solution space, and the cellular metabolites with turnover fluxes that were necessarily altered by the change in conditions increased from 118 to 271 of 1397. Availability: GIMMME has been implemented in Python and requires a COBRApy 0.2.x. The algorithm and sample data described here are freely available at: http://opencobra.sourceforge.net/

The purpose of this study was to test the effect of modulating the expression of the human low-density lipoprotein receptor (LDLr) in human embryonic kidney (293T) cells on Cyclosporin A (CsA) cellular uptake and CsA-mediated cytotoxicity. LDLr expression was modulated using RNA interference (RNAi) and an LDLr overexpression plasmid. One of the small-interfering RNA (siRNA) constructs, LDLr-792, showed a 60% decrease in LDLr protein expression. The downregulation effect was specific as transfection with an annexin V (AxV) siRNA construct did not decrease LDLr expression levels. AxV and ABCA1 expression levels were not affected in the cells transfected with LDLr-792 (LDLr(LOW) cells) compared to the controls. At a functional level, fluorescent low-density lipoprotein (LDL) (DiI-LDL) internalization in the LDLr(LOW) cells was decreased (30%) compared to control cells. We tested the dose-dependent cytotoxicity induced by CsA using a respiration assay. We found a decrease in CsA-mediated cytotoxicity in the range of CsA doses studied (1-10 microg/mL) in the LDLr(LOW) cells compared to the pSHAG-transfected cells, reaching a statistical significance at 10 microg/mL CsA. At higher CsA doses we found a significant decrease in LDLr expression. When the control and LDLr(LOW) cells were treated with another cytotoxic drug, gentamycin, there was no difference in the cell viability, suggesting that this effect is specific for CsA. We confirmed the association of LDLr expression levels with CsA uptake by overexpressing the LDLr. The LDLr overexpressing cells showed an enhanced uptake of radiolabelled CsA. Taken together these results suggest that CsA internalization and cytotoxicity are affected by the LDL receptor expression levels.

Purpose. Levels of TGF-β2 are higher in POAG aqueous humor, causing deposition of extracellular matrix (ECM) proteins, including fibronectin (FN), in the glaucomatous human trabecular meshwork (HTM) that may be responsible for elevated IOP. The purpose of this study was to identify the expression of cellular FN (cFN) isoforms (EDA and EDB) in HTM cells and tissues, and to determine whether TGF-β2 can induce cFN expression and fibril formation in cultured HTM cells. Methods. Expression of cFN mRNA isoforms and induction by recombinant TGF-β2 (5 ng/mL) were determined by quantitative RT-PCR. The TGF-β2 induction of EDA isoform protein expression and FN fibril formation were analyzed using Western immunoblots and immunocytochemistry (ICC), respectively. Immunohistochemistry (IHC) analysis was used to examine total FN and EDA isoform expression in normal (NTM) and glaucomatous (GTM) trabecular meshwork (TM) tissues. Results. Both cFN mRNA isoforms were expressed in cultured HTM cells and were induced by TGF-β2 after 2, 4, and 7 days (P < 0.05). Similarly, EDA isoform protein and fibril formation were increased after 4 and 7 days of TGF-β2 treatment. Finally, GTM tissues had significantly greater EDA isoform protein levels (1.7-fold, P < 0.05) compared to NTM tissues. Conclusions. This study demonstrated that cFN isoforms are expressed and induced in HTM cells by TGF-β2. Also, increased EDA isoform protein levels were seen in GTM tissues. Our findings suggest that induction of cFN isoform expression in the TM ECM may be a novel pathologic mechanism involved in the TM changes associated with glaucoma. PMID:24030464

The normal cellular prion protein (PrP(C)) plays an essential role in the development of prion diseases. Indirect evidence has suggested that different PrP(C) glycoforms may be expressed in different brain regions and perform distinct functions. However, due to a lack of monoclonal antibodies (Mabs) that are specific for mouse PrP(C), the expression of PrP(C) in the mouse brain has not been studied in great detail. We used Mabs specific for either the N-terminus or the C-terminus of the mouse PrP(C) to study its expression in the mouse brain by immunoblotting and immunohistochemistry. Immunoblotting studies demonstrated that the expression of PrP(C) differed quantitatively as well as qualitatively in different regions of the brain. The anti-C-terminus Mabs reacted with all three molecular weight bands of PrP(C); the anti-N-terminus Mabs only reacted with the 39-42 kDa PrP(C). The results from immunohistochemical staining revealed the spatial distribution of PrP(C) in the mouse brain, which were consistent with that from immunoblotting. Although expression of PrP(C) has been reported to be required for long-term survival of Purkinje cells, we were unable to detect PrP(C) in the Purkinje cell layer in the cerebellum with multiple anti-PrP Mabs. Our findings suggest that PrP(C) variants, i.e. various glycoforms and truncated forms, might be specifically expressed in different regions of mouse brain and might have different functions.

To better understand Euglena gracilis gene expression under different stress conditions (Chromium, Streptomycin or darkness), we undertook a survey of the E. gracilis transcriptome by cDNA sequencing and microarray analysis. First, we constructed a non-normalized cDNA library from the E. gracilis UTEX strain and sequenced a total of 1000 cDNAs. Six hundred and ten of these ESTs were similar to either Plantae or Protistae genes (e-valueexpression analysis indicated that 90 out of those 610 ESTs changed their expression level in response to different stress treatments (p<0.05). In addition, we detected 10 ESTs that changed expression levels irrespective of the tested stress. These may be considered as part of a larger set of stress-related genes in E. gracilis. Finally, we identified 23 unknown ESTs (U-ESTs) following the expression profiles of these putative stress-related genes suggesting that they could be related to the cellular mechanism of stress response.

This study was designed to determine changes in cell numbers, proliferation (using Ki-67) and EGFR expression in mouse bladder urothelium during the early and late radiation response. Groups of mice were irradiated with a single dose of 20 Gy and assayed 0-360 days later. Urothelial cells were counted. After immunohistochemistry, the absolute and relative numbers of Ki-67(+) and EGFR(+) cells were analyzed. Radiation exposure resulted in a decrease in total urothelial cell numbers to 49% by day 31, with restoration of cellularity by day 180. In contrast, at day 360, an increase in total cell number (143%) was seen. Slightly increased Ki-67 expression was found at days 120 and 180 after treatment, followed by a pronounced elevation at days 240 and 360. Compared to controls, higher EGFR expression was detected up to day 360 after irradiation. A positive correlation was found between total urothelial cells numbers and Ki-67 as well as EGFR expression. Radiation exposure results in an increased urothelial expression of EGFR that precedes urothelial restoration, indicating a contribution of the EGF/EGFR system to urothelial proliferation and differentiation. Further studies are needed to evaluate the impact of EGFR inhibition on radiation effects in the urinary bladder.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9), a gene exclusively expressed in the brain during embryonic stages but not in brains of adult mice, is an important cytoskeletal protein and regarded as a 'router/hub' in cellular signal transduction processes connecting external stimulation signals with downstream target proteins that can directly promote tumor metastasis. Numerous studies showed that NEDD9 has an essential role in cell proliferation, apoptosis, adhesion, migration and invasion. The roles of NEDD9, including the underlying mechanisms of its regulation of cell migration, its distinctive functions in various tumor stages and its association with other diseases, are required to be elucidated at large. Future studies of NEDD9 may provide a more profound understanding of the development of tumor invasiveness and NEDD9 may serve as a potential novel target for tumor therapy. The present review examined the significant roles of NEDD9 in the abovementioned processes.

The cellular slime mold Dictyostelium discoideum grows as unicellular free-living amoebae in the presence of nutrients. Upon starvation, the amoebae aggregate and form multicellular structures that each consist of a stalk and spores. D. discoideum encodes at least four proteins (Sir2A, Sir2B, Sir2C, and Sir2D) homologous to human SIRT. RT-PCR and WISH analyses showed that the genes for Sir2A, Sir2C, and Sir2D were expressed at high levels in growing cells but at decreased levels in developing cells, whereas the gene encoding Sir2B was expressed in the prestalk-cell region in the developmental phase.

In the standard cellular-automata model for a fault an element of stress is randomly added to a grid of boxes until a box has four elements, these are then redistributed to the adjacent boxes on the grid. The redistribution can result in one or more of these boxes having four or more elements in which case further redistributions are required. On the average added elements are lost from the edges of the grid. The model is modified so that the boxes have a scale-invariant distribution of sizes. The objective is to model a scale-invariant distribution of fault sizes. When a redistribution from a box occurs it is equivalent to a characteristic earthquake on the fault. A redistribution from a small box (a foreshock) can trigger an instability in a large box (the main shock). A redistribution from a large box always triggers many instabilities in the smaller boxes (aftershocks). The frequency-size statistics for both main shocks and aftershocks satisfy the Gutenberg-Richter relation with b = 0.835 for main shocks and b = 0.635 for aftershocks. Model foreshocks occur 28 percent of the time.

Power law scaling has been widely observed in the frequency distribution of landslide sizes. The exponent of the power-law characterizes the probability of landslide magnitudes and it thus represents an important parameter for hazard assessment. The reason for the universal scaling behavior of landslides is still debated and the role of topography has been explored in terms of possible explanation for this type of behavior. We built a simple cellular automata model to investigate this issue, as well as the relationships between the scaling properties of landslide areas and the changes suffered by the topographic surface affected by landslides. The dynamics of the model is controlled by a temporal rate of weakening, which drives the system to instability, and by topography, which defines both the quantity of the displaced mass and the direction of the movement. Results show that the model is capable of reproducing the scaling behavior of real landslide areas and suggest that topography is a good candidate to explain their scale-invariance. In the model, the values of the scaling exponents depend on how fast the system is driven to instability; they are less sensitive to the duration of the driving rate, thus suggesting that the probability of landslide areas could depend on the intensity of the triggering mechanism rather than on its duration, and on the topographic setting of the area. Topography preserves the information concerning the statistical distribution of areas of landslides caused by a driving mechanism of given intensity and duration.

Human T cell leukemia virus type I (HTLV-I) has been identified as the etiologic agent of adult T cell leukemia (ATL). HTLV-I encodes a transcriptional regulatory protein, Tax, which also functions as the viral transforming protein. Through interactions with a number of cellular transcription factors Tax can modulate cellular gene expression. Since the majority of Tax-responsive cellular genes are important regulators of cellular proliferation, the transactivating functions of Tax appear to be necessary for cellular transformation by HTLV-I. Gaining a complete understanding of the broad range of genes regulated by Tax, the temporal pattern of their expression, and their effects on cell function may identify early markers of disease progression mediated by this virus. PMID:11790876

The mevalonate pathway (MVP) and the anti-angiogenic effect of bisphosphonates have been shown to play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study determined the effect of the bisphosphonate, zoledronic acid and the replenishment of the MVP by geranylgeraniol on human gingival fibroblasts. Cell viability, apoptosis, morphological analysis using transmission electron microscopy, and gene expression for vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B, epiregulin and interferon-alpha were conducted. Results showed cellular viability was decreased in the presence of zoledronic acid and the co-addition of zoledronic acid with geranylgeraniol restored cell viability to control levels. Caspase 3/7 was detected in zoledronic-acid-treated cells indicating apoptosis. Transmission electron microscopy revealed dilation of the rough endoplasmic reticulum with zoledronic acid and the appearance of multiple lipid-like vesicles following the addition of geranylgeraniol. Zoledronic acid significantly (P < 0.05, FR > ± 2) up-regulated vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B and epiregulin at one or more time points but not interferon-alpha. Addition of geranylgeraniol resulted in a reduction in the expression of all five genes compared with zoledronic-acid-treated human gingival fibroblasts. The study concluded geranylgeraniol partially reversed the effects of zoledronic acid in human gingival fibroblasts both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the mevalonate pathway.

Over-expression of hexose transporters (Gluts), specifically Glut-1, is a common event in human malignancies. In prostate cancer (CaP), however, expression of Gluts has been characterized poorly. In this study, expression and distribution of Glut-1 and Glut-5 proteins were characterized using immunohistochemistry in 76 specimens of benign prostate, 10 specimens of high-grade intraepithelial neoplasia (HGPIN), and 28 specimens of CaP. In addition, mRNA expression of Glut-2, Glut-7, Glut-9, and Glut-11 was analyzed in a set of five specimens of benign prostate and CaP. In benign prostate, Glut-1 localized to the basal cells and to the basolateral membrane of secretory/luminal epithelial cells. Glut-5, however, localized to the apical membrane of secretory/luminal epithelial cells. In HGPIN, Glut-1 was immunohistochemically undetectable. Glut-5, however, localized to the apical membrane of the neoplastic epithelial cells. In CaP, Glut-1 and Glut-5, were immunohistochemically undetectable. However, over-expression of GLUT1 was observed in some specimens of highly proliferative intraductal CaP. Glut-7, Glut-9, and Glut-11 mRNAs were detected in benign prostate and CaP, however, only Glut-11 mRNA was consistently up-regulated in CaP compared to benign prostate. Low levels of expression of Glut-1 protein in the majority of CaP could explain, at least in part, the limited clinical applicability of positron emission tomography using 2-[18F]-fluoro-2-deoxy-D-glucose for imaging CaP. Moreover, expression of Glut-5 in HGPIN suggested that fructose could be utilized as potential metabolic substrate in HGPIN. Understanding the molecular mechanisms involved in regulation/dysregulation of Gluts in CaP could provide insight in the understanding of hexose metabolism in CaP.

Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity.

Background and Aims Previous studies suggest that low serum 25-hydroxyvitamin D [25(OH) D] levels are associated with reduced responsiveness to interferon and ribavirin therapy. We investigated the impact of vitamin D metabolites on HCV and cellular gene expression in cultured hepatoma cells. Methods HCV Replicon cell lines stably expressing luciferase reporter constructs (genotype 1b and 2a replicon) or JC1-Luc2a were incubated in the presence of vitamin D2, vitamin D3 or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Presence of HCV was quantified by a luciferase reporter assay and immunoblot of the Core protein. Synergy of interferon-alpha A/D (IFN-α) and 1,25(OH)2D3 was evaluated using the Chou-Talalay method. Cellular gene expression by microarray analysis using Illumina Bead Chips and real-time quantitative PCR. Results Vitamin D2, D3 and 1,25(OH)2D3 each demonstrated anti-HCV activity at low micro molar concentrations. In vitro conversion from D3 to 25(OH)D3 was shown by LC/MS/MS. Combination indices of 1,25(OH)2D3 and IFN-α demonstrated a synergistic effect (0.23-0.46) and significantly reduced core expression by immunoblot. Differentially expressed genes were identified between Huh7.5.1 cells in the presence and absence of 1,25(OH)2D3 and HCV. Genes involved with classical effects of vitamin D metabolism and excretion were activated, along with genes linked to autophagy such as G-protein coupled receptor 37 (GPR37) and Hypoxia-inducible factor 1-alpha (HIF1a). Additionally, additive effects of 1,25(OH)2D3 and IFN-α were seen on mRNA expression of chemokine motif ligand 20 (CCL20). Conclusions This study shows that vitamin D reduces HCV protein production in cell culture synergistically with IFN-α. Vitamin D also activates gene expression independently and additively with IFN-α and this may explain its ability to aid in the clearance of HCV in vivo. PMID:26594646

Background Among the vertebrates, teleost and urodele amphibians are capable of regenerating their central nervous system. We have used zebrafish as a model to study spinal cord injury and regeneration. Relatively little is known about the molecular mechanisms underlying spinal cord regeneration and information based on high density oligonucleotide microarray was not available. We have used a high density microarray to profile the temporal transcriptome dynamics during the entire phenomenon. Results A total of 3842 genes expressed differentially with significant fold changes during spinal cord regeneration. Cluster analysis revealed event specific dynamic expression of genes related to inflammation, cell death, cell migration, cell proliferation, neurogenesis, neural patterning and axonal regrowth. Spatio-temporal analysis of stat3 expression suggested its possible function in controlling inflammation and cell proliferation. Genes involved in neurogenesis and their dorso-ventral patterning (sox2 and dbx2) are differentially expressed. Injury induced cell proliferation is controlled by many cell cycle regulators and some are commonly expressed in regenerating fin, heart and retina. Expression pattern of certain pathway genes are identified for the first time during regeneration of spinal cord. Several genes involved in PNS regeneration in mammals like stat3, socs3, atf3, mmp9 and sox11 are upregulated in zebrafish SCI thus creating PNS like environment after injury. Conclusion Our study provides a comprehensive genetic blue print of diverse cellular response(s) during regeneration of zebrafish spinal cord. The data highlights the importance of different event specific gene expression that could be better understood and manipulated further to induce successful regeneration in mammals. PMID:24465396

The complex interactions of glomerular and tubular epithelial cells with the basal laminae play a critical role in renal function. Disruption of these interactions has been widely implicated in glomerular diseases and acute renal failure. MDC are a large family of membrane-bound proteins containing metalloprotease, disintegrin (integrin interaction sites), and cysteine-rich domains. Little information is available concerning the presence of MDC in the kidney or their role in renal pathophysiology. Using degenerate PCR primers for the conserved metalloprotease and disintegrin domains of this protein family, cDNA templates from tubules, whole glomeruli, and glomerular epithelial cells (GEC) yielded a single, 195-bp product, which on sequence analysis corresponded to a region in the disintegrin domain of MDC9. Northern analysis of poly(A)+ RNA from tubules, whole glomeruli, and GEC revealed a 3.9-kb transcript, identical to that of mouse MDC9. Using antibodies generated against a 21-amino acid peptide present in the metalloprotease domain of MDC9, Western analysis of concanavalin A-enriched glomerular microsomal extracts demonstrated both processed (76 kD) and unprocessed (116 kD) forms of MDC9, which upon reduction changed to the corresponding 84- and 124-kD forms. Histochemical studies revealed a basolateral localization of intrinsic MDC9 protein in renal cortical tubule cells and glomerular visceral epithelial cells, which colocalized with the beta1 integrin chain. Expression of green fluorescence protein MDC9 chimeric constructs in GEC or polarized Madin-Darby canine kidney epithelial cells revealed a similar punctate basolateral surface localization. Transient overexpression of the soluble disintegrin domain-green fluorescence protein chimera in GEC led to dramatic changes in cellular morphology with rounding and detachment from cell monolayers. These studies document the presence of MDC9 in renal epithelial cells and suggest an important role for MDC9 in renal

Proliferation of stem/progenitor cells during development provides for the generation of mature cell types in the CNS. While adult brain proliferation is highly restricted in the mammals, it is widespread in teleosts. The extent of adult neural proliferation in the weakly electric fish, Gymnotus omarorum has not yet been described. To address this, we used double thymidine analog pulse-chase labeling of proliferating cells to identify brain proliferation zones, characterize their cellular composition, and analyze the fate of newborn cells in adult G. omarorum. Short thymidine analog chase periods revealed the ubiquitous distribution of adult brain proliferation, similar to other teleosts, particularly Apteronotus leptorhynchus. Proliferating cells were abundant at the ventricular-subventricular lining of the ventricular-cisternal system, adjacent to the telencephalic subpallium, the diencephalic preoptic region and hypothalamus, and the mesencephalic tectum opticum and torus semicircularis. Extraventricular proliferation zones, located distant from the ventricular-cisternal system surface, were found in all divisions of the rombencephalic cerebellum. We also report a new adult proliferation zone at the caudal-lateral border of the electrosensory lateral line lobe. All proliferation zones showed a heterogeneous cellular composition. The use of short (24 h) and long (30 day) chase periods revealed abundant fast cycling cells (potentially intermediate amplifiers), sparse slow cycling (potentially stem) cells, cells that appear to have entered a quiescent state, and cells that might correspond to migrating newborn neural cells. Their abundance and migration distance differed among proliferation zones: greater numbers and longer range and/or pace of migrating cells were associated with subpallial and cerebellar proliferation zones. PMID:25249943

There is a clear link between overweight, gain of white adipose tissue, and diabetes type 2 (T2D). The molecular mechanism of the gain of adipose tissue is linked with the expression of high mobility group protein AT-hook 2 (HMGA2), and recent studies revealed an association with a SNP near HMGA2. In this study, we investigated the gene expression of HMGA2, p14 (Arf) , CDKN1A, and BAX in human abdominal subcutaneous white adipose tissue from 157 patients. We found a significant higher HMGA2 expression in obese individuals than in non-obese patients. Furthermore, the HMGA2 expression in white adipose tissue in patient with type 2 diabetes was significantly higher than in nondiabetic patients. There is an association between the DNA-binding nonhistone protein HMGA2 and the risk of developing T2D that remains mechanistically unexplained so far. Likewise, p14(Arf), an inducer of cellular senescence, has been associated with the occurrence of T2D. The data of the present study provide evidence that both proteins act within the same network to drive proliferation of adipose tissue stem and precursor cells, senescence, and increased risk of T2D, respectively.

Recently, the second mammalian chitinase, designated acidic mammalian chitinase (AMCase), has been identified in human, mouse, and cow. In contrast to the earlier identified macrophage-derived chitinase (chitotriosidase), this chitinase is richly expressed in the gastrointestinal (GI) tract, suggesting its role in digestion of chitin-containing foods as well as defense against chitin-coated microorganisms and parasites. This in situ hybridization study first revealed cellular localization of the gut-type chitinase in the mouse and chicken. In adult mice, the parotid gland, von Ebner's gland, and gastric chief cells, all of which are exocrine cells of the serous type, expressed the gut chitinase mRNA. In the chicken, oxyntico-peptic cells in glandular stomach (proventriculus) and hepatocytes expressed the chitinase mRNA. Because cattle produce the gut chitinase (chitin-binding protein b04) only in the liver, the gut chitinases in mammals and birds have three major sources of production, i.e., the salivary gland, stomach, and liver. During ontogenetic development, the expression level in the parotid gland and stomach of mice increased to the adult level before weaning, whereas in the stomach of chickens intense signals were detectable in embryos from incubation day 7.

Obscurin and obscurin-associated kinase are two products of the obscurin transcriptional unit that encodes a recently identified giant muscle-specific protein obscurin. In this study, we characterized the developmental expression and cellular localization of obscurin and obscurin-associated kinase in cardiac muscle cells. We cloned murine obscurin-associated kinase and found that it is abundantly expressed in the heart as two isotypes encoded by 2.2 and 4.9 kb sequences. The 2.2 kb isotype of the kinase was more prominently expressed than the 4.9 kb isotype. Both obscurin and the kinase-like domains were progressively upregulated since the early stages of cardiac development. Obscurin-associated kinase was expressed at higher levels than obscurin at early stages of cardiomyogenesis. Increasing intensity of obscurin expression in the developing heart positively correlated with progressive cell differentiation and was higher in the ventricles compared to the atria. These data were supported by the results of experiments with primary cardiac cell cultures. Obscurin localization changed from a weakly immunopositive diffuse pattern in poorly differentiated cells to an intensely immunolabeled cross-striated distribution at the level of mid-A-bands and Z-disks during the assembly of the myofibrillar contractile apparatus. In dividing myocytes, unlike the interphase cells, obscurin translocated from disassembling myofibrils into a diffuse granulated pattern segregated separately from alpha-actinin-immunopositive aggregates. Obscurin-associated kinase was localized mainly to cell nuclei with increasing incorporation into the Z-disks during differentiation. Our results suggest that these two novel proteins are involved in the progression of cardiac myogenesis during the transition to advanced stages of heart development. 2007 Wiley-Liss, Inc.

Injury to the energy-storing superficial digital flexor tendon is common in equine athletes and is age-related. Tenocytes in the superficial digital flexor tendon of adult horses appear to have limited ability to respond adaptively to exercise or prevent the accumulation of strain-induced microdamage. It has been suggested that conditioning exercise should be introduced during the growth period, when tenocytes may be more responsive to increased quantities or intensities of mechanical strain. Tenocytes are linked into networks by gap junctions that allow coordination of synthetic activity and facilitate strain-induced collagen synthesis. We hypothesised that there are reductions in cellularexpression of the gap junction proteins connexin (Cx) 43 and 32 during maturation and ageing of the superficial digital flexor tendon that do not occur in the non-injury-prone common digital extensor tendon. Cryosections from the superficial digital flexor tendon and common digital extensor tendon of 5 fetuses, 5 foals (1–6 months), 5 young adults (2–7 years) and 5 old horses (18–33 years) were immunofluorescently labelled and quantitative confocal laser microscopy was performed. Expression of Cx43 and Cx32 protein per tenocyte was significantly higher in the fetal group compared with all other age groups in both tendons. The density of tenocytes was found to be highest in immature tissue. Higher levels of cellularity and connexin protein expression in immature tendons are likely to relate to requirements for tissue remodelling and growth. However, if further studies demonstrate that this correlates with greater gap junctional communication efficiency and synthetic responsiveness to mechanical strain in immature compared with adult tendons, it could support the concept of early introduction of controlled exercise as a means of increasing resistance to later injury. PMID:17848160

Fourteen members of the monocarboxylate transporter (MCT, SLC16) family have been identified, each having a different tissue distribution and substrate specificity. The expression of monocarboxylate transporters MCT1 and MCT4 have been studied in the gastrointestinal tract of ruminants; however, details of the expression of other MCT isoforms in the various parts of ruminant gastrointestinal tract are lacking. Reverse transcription with the polymerase chain reaction was used to study the regional distribution of MCT2, MCT3, and MCT5-MCT14 in the cattle gastrointestinal tract and verified the existence of MCT mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT9, MCT10, MCT13, and MCT14 in the ruminal and abomasal epithelia, mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT10, MCT13, and MCT14 in the jejunum, and mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT13, and MCT14 in the caecum of cattle. At the cellular level, immunohistochemical studies localized MCT2, MCT7, and MCT8 proteins in the cattle rumen, abomasum, jejunum, and caecum. This is the first study to detect the expression of various MCT isoforms in the gastrointestinal tract of a ruminant species. Our data suggest that these transporter proteins are involved in essential physiologic processes and are possible molecular targets for studying the regulation of the transport of short-chain monocarboxylates, aromatic amino acids, and thyroid hormones across the gastrointestinal tract of cattle.

Background The activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised. Methodology/Principal Findings We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy. Conclusions/Significance We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular

TFF3 is a member of the trefoil factor family (TFF) predominantly secreted by mucous epithelia. Minute amounts are also expressed in the immune system and the brain. In the latter, particularly the hypothalamo-pituitary axis has been investigated in detail in the past. Functionally, cerebral TFF3 has been reported to be involved in several processes such as fear, depression, learning and object recognition, and opiate addiction. Furthermore, TFF3 has been linked with neurodegenerative and neuropsychiatric disorders (e.g., Alzheimer's disease, schizophrenia, and alcoholism). Here, using immunohistochemistry, a systematic survey of the TFF3 localization in the adult human brain is presented focusing on extrahypothalamic brain areas. In addition, the distribution of TFF3 in the developing human brain is described. Taken together, neurons were identified as the predominant cell type to express TFF3, but to different extent; TFF3 was particularly enriched in various midbrain and brain stem nuclei. Besides, TFF3 immunostaining staining was observed in oligodendroglia and the choroid plexus epithelium. The wide cerebral distribution should help to explain its multiple effects in the CNS.

Novel techniques for analyzing microarray data are constantly being developed. Though many of the methods contribute to biological discoveries, inability to properly evaluate the novel techniques limits their ability to advance science. Because the underlying distribution of microarray data is unknown, novel methods are typically tested against the assumed normal distribution. However, microarray data are not, in fact, normally distributed, and assuming so can have misleading consequences. Using an Affymetrix technical replicate spike-in data set, we show that oligonucleotide expression values are not normally distributed for any of the standard methods for calculating expression values. The resulting data tend to have a large proportion of skew and heavy tailed genes. Additionally, we show that standard methods can give unexpected and misleading results when the data are not well approximated by the normal distribution. Robust methods are therefore recommended when analyzing microarray data. Additionally, new techniques should be evaluated with skewed and/or heavy-tailed data distributions.

Hat1 was the first histone acetyltransferase identified, however its biological function is still unclear. In this report, we show that the human Hat1 has two isoforms. Isoform a has 418 amino acids (aa) and is localized exclusively in the nuclear matrix of normal human keratinocytes (NHKs). Isoform b has 334 aa and is located in thecytoplasm, the nucleoplasm, attached to the chromatin and to the nuclear matrix. Immunohistochemical analyses revealed that the bulk of Hat1 is confined to the nucleus, with much lesser amounts in the cytoplasm. Cells undergoing mitotic division have an elevated amount of Hat1 compared to non-mitotic ones. NHKs exposed to hydrogen peroxide (H2O2) or to a beam of high mass and energy (HZE) ion particles expressed bright nuclear staining for Hat1, a phenotype that was not observed in NHKs exposed to &947;-rays. We established that the enhanced nuclear staining for Hat1 in response to these treatments is regulated by the PI3K and the MAPK signaling pathways. Our observations clearly implicate Hat1 in the cellular response assuring the survival of the treated cells.

Changes in cellular calcium (Ca(2+)) handling are thought to underlie the altered contraction that occurs during cardiac hypertrophy and failure. Recent work has highlighted the importance of t-tubules in the control of intracellular Ca(2+). The present study was performed to investigate whether changes in the distribution of I (Ca) between the surface and t-tubule membranes might contribute to the altered Ca(2+) handling observed during compensated hypertrophy in the spontaneously hypertensive rat (SHR). Experiments were performed on ventricular myocytes isolated from 5-month-old SHR and normotensive Wistar-Kyoto (WKY) control rats. Osmotic shock using formamide was used to disrupt the t-tubular system and the whole-cell patch clamp technique used to monitor I (Ca) in the presence and absence of t-tubules. Membrane capacitance and I (Ca) were greater in control SHR than WKY myocytes; following detubulation, cell capacitance and I (Ca) both decreased and were no longer significantly different in the two cell types. The density of I (Ca) was not significantly different in control SHR and WKY cells or in detubulated myocytes from the two species. These data suggest that the distribution of I (Ca) is unchanged in SHR myocytes compared to WKY controls; I (Ca) density in the t-tubules was 1.2-fold greater than in the sarcolemma in both strains. These data also imply that the increase in surface area in SHR myocytes is due principally to an increase in t-tubular area, which is accompanied by an approximately equivalent increase in I (Ca), so that the density of I (Ca) at the cell surface and in the t-tubules remains the same. These changes would be expected to retain cell function and synchronicity of Ca(2+) release in the SHR at this stage of compensated hypertrophy.

Dystrophin Dp40 is the shortest protein encoded by the DMD (Duchenne muscular dystrophy) gene. This protein is unique since it lacks the C-terminal end of dystrophins. In this data article, we describe the subcellular localization, nuclear export signals and the three-dimensional structure modeling of putative Dp40 proteins using bioinformatics tools. The Dp40 wild type protein was predicted as a cytoplasmic protein while the Dp40n4 was predicted to be nuclear. Changes L93P and L170P are involved in the nuclear localization of Dp40n4 protein. A close analysis of Dp40 protein scored that amino acids 93LEQEHNNLV101 and 168LLLHDSIQI176 could function as NES sequences and the scores are lost in Dp40n4. In addition, the changes L93/170P modify the tertiary structure of putative Dp40 mutants. The analysis showed that changes of residues 93 and 170 from leucine to proline allow the nuclear localization of Dp40 proteins. The data described here are related to the research article entitled “EF-hand domains are involved in the differential cellulardistribution of dystrophin Dp40” (J. Aragón et al. Neurosci. Lett. 600 (2015) 115–120) [1]. PMID:26217814

The cellular prion protein (PrP), a cell membrane-bound glycoprotein mainly located in the dendrites and axons of the central nervous system (CNS), is responsible for transmissible spongiform encephalopathies in mammals. PrP genes are widely conserved in vertebrates. In birds, the presence of PrP mRNA has been confirmed in neurons of the chicken brain, but localization of the protein remains to be determined. In the present study, we demonstrated the regional distribution of PrP in the CNS of adult chickens by immunohistochemical staining with a monoclonal antibody that recognizes chicken PrP 161-164. Immunoreactivity was observed in the neuropil, but not in neuronal somata or glial cells. It was preferentially intense in the olfactory bulb, the dorsal thalamus, the hypothalamus, and most regions of the telencephalon. Immunostaining became less intense toward the brainstem, but many nuclei were immunoreactive. Among brainstem nuclei, moderate immunostaining was observed in the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, and substantia gelatinosa Rolandi. The cerebellar cortex was devoid of PrP immunoreactivity. The dorsal horn in the spinal cord was strongly immunoreactive. In situ hybridization with two probes of the C-terminal portion demonstrated localization of PrP mRNA in neurons of the brain and spinal cord. These findings suggest that PrP in the chicken CNS is localized in the dendrites and axons of neurons and that it is associated with certain sensory systems.

In comparison with controls neutrophil granulocytes from Rheumatoid arthritis (RA), Infantile Neuronal Ceroid Lipofuscinosis (INCL), Chronic Lymphatic Leukemia (L) and Aplastic Anemia (AA) displayed significant alterations in essential and non-essential elements which might be interpreted as fingerprints of these deseases. The neutrophils from RA patients displayed alterations in the concentrations of iron, calcium, strontium, manganese, zinc and copper. INCL displayed alterations in the concentrations of iron and copper but in the INCL disease the iron concentration was about 2 times higher than in RA. In leukemia, aluminium was observed but not in the controls (< 0.5 μg/ g). The zinc concentration was lowered in leukemia. Aplastic anemia displayed alterations in zirconium, arsenic, molybdenum, iron and zinc. The platelets from RA, INCL, L and AA patients also displayed alterations in the elemental profiles. The platelets from AA patients displayed a unique elemental distribution of arsenic, zirconium and molybdenum. The elemental profiles of the thrombocytes and neutrophils might be used as a complement in the diagnosis of the examined diseases and in therapy the elemental profile might be used to monitor drugs at the cellular level.

This study was carried out to resolve the discrepancy of data for the proportion of ascorbic acid and dehydroascorbic acid in persimmon leaves at the final stage of the season and to clarify their cellulardistributions using histochemical and biochemical techniques. Fresh persimmon leaves were collected and used on July 31, September 5 and October 7, 1996. Ascorbic acid and dehydroascorbic acid in subcellular fractions were determined by the HPLC method that was found to be the most reliable for separation. The percent of dehydroascorbic acid in the total leaves was found to be almost constant (between 32 and 37%) in all preparations tested. In all preparations, more than 90% of the ascorbic acid and dehydroascorbic acid was found in the soluble fraction. The histochemical detection of ascorbic acid and an electron micrograph of persimmon leaf cells showed that the reactive color, after the reduction of silver nitrate under acidic conditions, in the leaves of all three preparations was mainly found on the face side of columned-type palisade parenchyma cells where chloroplasts were not rich and large vacuoles were seen. On the inner side of the palisade parenchyma cells where chloroplasts were the richest, only weak color development was observed. This study demonstrates that the percent of dehydroascorbic acid in persimmon leaves did not exceed 40% at least until October 7. It also shows that in persimmon leaf cells, ascorbic acid is mainly localized in the cytosol of palisade parenchyma tissue cells where large vacuoles are seen.

Efficient delivery of copper ions to specific intracellular targets requires copper chaperones that acquire metal cargo through unknown mechanisms. Here we demonstrate that the human and yeast copper chaperones (CCS) for superoxide dismutase 1 (SOD1), long thought to exclusively reside in the cytosol and mitochondrial intermembrane space, can engage negatively charged bilayers through a positively charged lipid-binding interface. The significance of this membrane-binding interface is established through SOD1 activity and genetic complementation studies in Saccharomyces cerevisiae, showing that recruitment of CCS to the membrane is required for activation of SOD1. Moreover, we show that a CCS:SOD1 complex binds to bilayers in vitro and that CCS can interact with human high affinity copper transporter 1. Shifting current paradigms, we propose that CCS-dependent copper acquisition and distribution largely occur at membrane interfaces and that this emerging role of the bilayer may reflect a general mechanistic aspect of cellular transition metal ion acquisition. PMID:24297923

Cell-based immunotherapies are among the most promising approaches for developing effective and targeted immune response. However, their clinical usefulness and the evaluation of their efficacy rely heavily on complex quality control assessment. Therefore, rapid systematic methods are urgently needed for the in-depth characterization of relevant factors affecting newly developed cell product consistency and the identification of reliable markers for quality control. Using dendritic cells (DCs) as a model, we present a strategy to comprehensively characterize manufactured cellular products in order to define factors affecting their variability, quality and function. After generating clinical grade human monocyte-derived mature DCs (mDCs), we tested by gene expression profiling the degrees of product consistency related to the manufacturing process and variability due to intra- and interdonor factors, and how each factor affects single gene variation. Then, by calculating for each gene an index of variation we selected candidate markers for identity testing, and defined a set of genes that may be useful comparability and potency markers. Subsequently, we confirmed the observed gene index of variation in a larger clinical data set. In conclusion, using high-throughput technology we developed a method for the characterization of cellular therapies and the discovery of novel candidate quality assurance markers. PMID:23147403

The effects of extracellular zinc concentration on metallothionein gene expression and cellular zinc accumulation were studied in IRD-98 cells. This is a non-transformed clonal line established by Negrel, et al. from fetal rat small intestine which possess characteristics of small bowel epithelial cells. Cells were maintained in DMEM and grown to confluent monolayers. The response to media zinc concentrations over the range of 5-150 {mu}mol/L was assessed. After 24 h in culture, cell zinc and metallothionein protein concentrations were significantly increased in cells provided higher levels of media zinc. Subsequent time course experiments showed that cells exposed to higher zinc levels had significant elevations in both metallothionein mRNA, peaking at 24 h, and metallothionein protein increasing through 48 h. Furthermore, cell zinc concentrations were significantly increased. At 48 h of culture, greater than 50% of the additional cellular zinc accumulated could be attributed to elevated metallothionein protein levels. These cells represent a zinc-responsive model to examine the mechanism of zinc uptake and transcellular transport by intestinal cells and the regulatory factors involved.

ABSTRACT Cystine and cysteine are important molecules for pathways such as redox signaling and regulation, and thus identifying cellular deficits upon deletion of the Saccharomyces cerevisiae cystine transporter Ers1p allows for a further understanding of cystine homeostasis. Previous complementation studies using the human ortholog suggest yeast Ers1p is a cystine transporter. Human CTNS encodes the protein Cystinosin, a cystine transporter that is embedded in the lysosomal membrane and facilitates the export of cystine from the lysosome. When CTNS is mutated, cystine transport is disrupted, leading to cystine accumulation, the diagnostic hallmark of the lysosomal storage disorder cystinosis. Here, we provide biochemical evidence for Ers1p-dependent cystine transport. However, the accumulation of intracellular cystine is not observed when the ERS1 gene is deleted from ers1-Δ yeast, supporting the existence of modifier genes that provide a mechanism in ers1-Δ yeast that prevents or corrects cystine accumulation. Upon comparison of the transcriptomes of isogenic ERS1+ and ers1-Δ strains of S. cerevisiae by DNA microarray followed by targeted qPCR, sixteen genes were identified as being differentially expressed between the two genotypes. Genes that encode proteins functioning in sulfur regulation, cellular respiration, and general transport were enriched in our screen, demonstrating pleiotropic effects of ers1-Δ. These results give insight into yeast cystine regulation and the multiple, seemingly distal, pathways that involve proper cystine recycling. PMID:27142334

Background: The up-regulation of galectin-3, galectin-9, and galectin-15 expression in the luminal and glandular epithelium was reported in preparation of the endometrium for embryo implantation at the midlutheal phase. However, no data was available regarding the expression and the distribution pattern of galectin-8 in the human endometrium during a regular menstrual cycle. Objective: The current study designed to investigate the expression and the distribution pattern of galectin-8, a beta-galactoside-binding lectin in the human endometrium during both proliferative and luteal phases of a regular menstrual cycle. Materials and Methods: Endometrial biopsies were obtained from the anterior wall of the uterine cavity of 16 women (proliferative phase: n=4, lutheal phase: n=12). All female patients with mean age of 37.5 years were fertile (range 25-45). Each biopsy was divided into three pieces; one piece was fixed in formaldehyde for light microscopy and immunohistochemistry. The second portion fixed in glutaraldehyde for scanning electron microscopy and the third portion was prepared for western blot analysis. Results: Data of immunoblotting revealed a molecular weight of 34 kD band with high intensity in the lutheal phase samples. The immunohistochemistry staining demonstrated that galectin-8 expressed at a very low concentration during the proliferative phase, but showed a high expression throughout the lutheal phase. The expression of galectin-8 observed in luminal surface epithelium, glandular epithelium and stroma. Conclusion: The up-regulation of the expression of galectin-8 during lutheal phase may suggest galectin-8 as one of the potential molecular marker of the endometrial receptivity. These data propose that galectin-8 may play an important role during the initial events of human embryo implantation. PMID:24639695

Intracellular processes rarely work in isolation but continually interact with the rest of the cell. In microbes, for example, we now know that gene expression across the whole genome typically changes with growth rate. The mechanisms driving such global regulation, however, are not well understood. Here we consider three trade-offs that, because of limitations in levels of cellular energy, free ribosomes, and proteins, are faced by all living cells and we construct a mechanistic model that comprises these trade-offs. Our model couples gene expression with growth rate and growth rate with a growing population of cells. We show that the model recovers Monod’s law for the growth of microbes and two other empirical relationships connecting growth rate to the mass fraction of ribosomes. Further, we can explain growth-related effects in dosage compensation by paralogs and predict host–circuit interactions in synthetic biology. Simulating competitions between strains, we find that the regulation of metabolic pathways may have evolved not to match expression of enzymes to levels of extracellular substrates in changing environments but rather to balance a trade-off between exploiting one type of nutrient over another. Although coarse-grained, the trade-offs that the model embodies are fundamental, and, as such, our modeling framework has potentially wide application, including in both biotechnology and medicine. PMID:25695966

Myosin VI is a motor protein which is necessary for the morphogenesis of epithelial tissues during Drosophila development. The spatial and temporal expression of Myosin VI was examined by expressing a GFP (Green Fluorescent Protein) tagged Myosin VI molecule (PGM), under the control of a Myosin VI-Gal4 line. PGM was present in tissues that were shown previously to express Myosin VI, such as the ovarian follicle epithelium, and the individualization complex; and in other tissues, including the trachea, the midgut, the salivary glands and the imaginal discs. The GFP-tagged Myosin V1 rescued the male sterile phenotype of Jaguar showing it is functional in vivo. Within individual cells, the role of the head and neck domain and the tail domain in targeting of the Myosin V1 molecule was examined by investigating the localisation of the separate domains tagged to GFP. In salivary glands and follicle cells the head and neck domains were concentrated in the cell nucleus, where the minus end of each actin filament is located. We found that the tail domain anchors the whole molecule outside of the nucleus. Similarly, in the individualization complex in the testes, the tail anchors the whole molecule to the base of the complex while the separated head with neck domain becomes scattered along the entire actin molecule suggesting the cellular location may be determined by cargo proteins that bind to the tail domain rather than by the movement of Myosin VI along the actin filaments.

Interaction of human papilloma virus oncoproteins E6 and E7 with cell cycle proteins leads to disturbances of the cell cycle mechanism and subsequent alteration in the expression of some proteins, such as p16INK4a, cyclin D1, p53 and KI67. In this study, we compared alterations in the expression of these proteins during several stages of intraepithelial cervical carcinogenesis. Accordingly, an immunohistochemical study was performed on 50 cervical biopsies, including negative cases and intraepithelial neoplasias. The expression patterns of these markers were correlated with the histopathological diagnosis and infection with HPV. The p16INK4a, followed by Ki67, showed better correlation with cancer progression than p53 and cyclin D1, which recommends their use in the evaluation of cervical carcinogenesis. These monoclonal antibodies can be applied to cervical biopsy specimens to identify lesions transformed by oncogenic HPV, separating CIN 1 (p16INK4a positive) and identifying high-grade lesions by an increase in the cellular proliferation index (Ki67). In this way, we propose immunomarkers that can be applied in clinical practice to separate patients who need a conservative therapeutic approach from those who require a more aggressive treatment.

The c-myc regulatory region includes binding sites for a large set of transcription factors. The present studies demonstrate that in the absence of FBP [far upstream element (FUSE)-binding protein], which binds to the single-stranded FUSE, the remainder of the set fails to sustain endogenous c-myc expression. A dominant-negative FBP DNA-binding domain lacking effector activity or an antisense FBP RNA, expressed via replication-defective adenovirus vectors, arrested cellular proliferation and extinguished native c-myc transcription from the P1 and P2 promoters. The dominant-negative FBP initially augmented the single-stranded character of FUSE; however, once c-myc expression was abolished, melting at FUSE could no longer be supported. In contrast, with antisense FBP RNA, the single-stranded character of FUSE decreased monotonically as the transcription of endogenous c-myc declined. Because transcription is the major source of super-coiling in vivo, we propose that by binding torsionally strained DNA, FBP measures promoter activity directly. We also show that FUSE is predicted to behave as a torsion-regulated switch poised to regulate c-myc and to confer a higher order regulation on a large repertoire of factors. PMID:10698944

Myotendinous strain injury is the most common injury of human skeletal muscles because the majority of muscle forces are transmitted through this region. Although the immediate response to strain injury is well characterized, the chronic response to myotendinous strain injury is less clear. Here we examined the molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a microdystrophin transgene (microdystrophinΔR4–R23). We found that muscles with myotendinous strain injury had an increased expression of utrophin and α7-integrin together with the dramatic restructuring of peripheral myofibrils into concentric rings. The sarcolemma of the microdystrophinΔR4–R23/mdx gastrocnemius muscles was highly protected from experimental lengthening contractions, better than wild-type muscles. We also found a positive correlation between myotendinous strain injury and ringed fibers in the HSALR (human skeletal actin, long repeat) mouse model of myotonic dystrophy. We suggest that changes in protein expression and the formation of rings are adaptations to myotendinous strain injury that help to prevent muscle necrosis and retain the function of necessary muscles during injury, ageing and disease. PMID:18799475

Myotendinous strain injury is the most common injury of human skeletal muscles because the majority of muscle forces are transmitted through this region. Although the immediate response to strain injury is well characterized, the chronic response to myotendinous strain injury is less clear. Here we examined the molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a microdystrophin transgene (microdystrophin(DeltaR4-R23)). We found that muscles with myotendinous strain injury had an increased expression of utrophin and alpha7-integrin together with the dramatic restructuring of peripheral myofibrils into concentric rings. The sarcolemma of the microdystrophin(DeltaR4-R23)/mdx gastrocnemius muscles was highly protected from experimental lengthening contractions, better than wild-type muscles. We also found a positive correlation between myotendinous strain injury and ringed fibers in the HSA(LR) (human skeletal actin, long repeat) mouse model of myotonic dystrophy. We suggest that changes in protein expression and the formation of rings are adaptations to myotendinous strain injury that help to prevent muscle necrosis and retain the function of necessary muscles during injury, ageing and disease.

The phenomenon of oscillations in probability distribution functions of number of components is found for a model of stochastic gene expression. It takes place in cases of low levels of molecules or strong intracellular noise. The oscillations distinguish between more probable even and less probable odd number of particles. The even-odd symmetry restores as the number of molecules increases with the probability distribution function tending to Poisson distribution. We discuss the possibility of observation of the phenomenon in gene, protein, and mRNA expression experiments.

The phenomenon of oscillations in probability distribution functions of number of components is found for a model of stochastic gene expression. It takes place in cases of low levels of molecules or strong intracellular noise. The oscillations distinguish between more probable even and less probable odd number of particles. The even-odd symmetry restores as the number of molecules increases with the probability distribution function tending to Poisson distribution. We discuss the possibility of observation of the phenomenon in gene, protein, and mRNA expression experiments.

The cryosurvival of sperm requires cell signaling mechanisms to adapt to anisotonic conditions during the freezing and thawing process. Chaperone proteins heat shock protein 70 (HSP 70) and heat shock protein 90 (HSP 90; recently renamed HSPA and HSPC, respectively) facilitate some of these cell signaling events in somatic cells. Sperm were evaluated for their cellularexpression and levels of phosphorylation of both HSP 70 and HSP 90 under anisotonic conditions as a potential model for cell signaling during the cryopreservation of macaque spermatozoa. In order to monitor the level of stress, the motility and viability parameters were evaluated at various time points. Cells were then either prepared for phosphoprotein enrichment or indirect immunocytochemistry. As controls, the phosphoserine, phosphothreonine, and phosphotyrosine levels were measured under capacitation and cryopreservation conditions and were compared with the phosphoprotein levels expressed under osmotic conditions. As expected, there was an increase in the level of tyrosine phosphorylation under capacitation and cryopreservation conditions. There was also a significant increase in the level of all phosphoproteins under hyperosmotic conditions. There was no change in the level of expression of HSP 70 or 90 under osmotic stress conditions as measured by Western blot. The enrichment of phosphoproteins followed by Western immunoblotting revealed an increase in the phosphorylation of HSP 70 but not HSP 90 under osmotic stress conditions. Indirect immunofluorescence localized HSP 70 to the postacrosomal region of sperm, and the level of membrane expression of HSP 70 was significantly affected by anisotonic conditions, as measured by flow cytometry. Taken together, these results suggest a differential role for HSP 70 and HSP 90 during osmotic stress conditions in rhesus macaque sperm.

We have examined the pattern of differentiation of the small intestinal epithelium in fetal rats during the 17th through 21st days of gestation. Five genes expressed in late fetal, neonatal, and adult enterocytes were used as markers of differentiation. They encode three homologous small cytoplasmic hydrophobic ligand binding proteins--liver fatty acid binding protein (L-FABP), intestinal fatty acid binding protein (I-FABP), and cellular retinol binding protein II (CRBP II)--and two apolipoproteins--apoAI and apoAIV. RNA blot hybridization studies indicated that gradients in mRNA concentration from the proximal small intestine to colon appear coincident with the initiation of rapid epithelial cell proliferation and villus formation (days 17-19 of the 22-day gestation period). Immunocytochemical studies disclosed a remarkably heterogeneous pattern of cell-specific expression of the three hydrophobic ligand binding proteins that was not apparent with either apoAIV or apoAI. This "mosaic" staining pattern was observed in morphologically similar cells occupying identical topographic positions along nascent villi in 17- to 18-day fetuses. The onset and resolution of this mosaicism varies between I-FABP, L-FABP, and CRBP II in the proximal small bowel, although it completely resolves by the first postnatal day. The distal small intestine exhibits a developmental delay of 1-2 days in the appearance of this heterogeneous pattern of initial gene expression. Double-label immunofluorescent analyses using L-FABP and I-FABP antibodies indicated that on the 18th day of gestation the proximal small intestinal columnar epithelium contains several populations of enterocytes expressing neither, one, or both proteins. The potential significance of this mosaic pattern of intestinal epithelial differentiation is discussed in light of recent studies with transgenic and chimeric mice.

A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2Apro) under the control of tetracycline has been obtained. Synthesis of 2Apro induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2Apro cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2Apro, prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2Apro still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2Apro. Moreover, synthesis of 2Apro in 2A7d cells complements the translational defect of a poliovirus 2Apro-defective variant. These results show that poliovirus 2Apro expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2Apro functions, to complement poliovirus 2Apro mutants, and to test antiviral compounds. PMID:10666269

Prion diseases are believed to develop from the conformational change of normal cellular prion protein (PrPc) to a pathogenic isoform (PrPsc). PrPc is present in both the central nervous system and many peripheral tissues, although protein concentration is significantly lower in non-neuronal tissues. PrPc expression is essential for internalization and replication of the infectious agent. Several works have pointed to the gastrointestinal (GI) tract as the principal site of entry of PrPsc, but how passage through the GI mucosa occurs is not yet known. Here we studied PrPc expression using Western blot, RT-PCR, and immunohistochemistry in rat GI tract. PrPc mRNA and protein were detected in corpus, antrum, duodenum, and colon. Immunoreactivity was found in scattered cells of the GI epithelium. With double immunofluorescence, these cells have been identified as neuroendocrine cells. PrPc immunostaining was found in subsets of histamine, somatostatin (Som), ghrelin, gastrin (G), and serotonin (5HT) cells in stomach. In small and large bowel, PrPc cells co-localized with subpopulations of 5HT-, Som-, G-, and peptide YY-immunolabeled cells. Our results provide evidence for a possible and important role of endocrine cells in the internalization of PrPsc from gut lumen. Copyright The Histochemical Society, Inc.

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. We propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.

The tumor suppressor p53 plays a central role in the regulation of cellular growth and apoptosis. In Saccharomyces cerevisiae, over-expression of the human wtp53 leads to growth inhibition and cell death on minimal medium. In the present work, we showed that deletion of the nuclear localization signal (NLSI) of p53 restores the yeast growth. In this heterologous context, the level of p53∆NLSI was low and the protein mainly located in the cytoplasm while the wtp53 was observed in both the cytoplasmic and nuclear compartments. Interestingly, the wtp53 protein was observed in the mitochondria, whereas the p53∆NLSI protein failed to localize in mitochondria. Moreover, mitochondrial morphology defect and release of cytochrome c in the cytosol were noticed only in the yeast strain expressing the wtp53. In conclusion, our results provide evidence that the human wtp53 is active in S. cerevisiae probably through dependent and independent transcriptional mechanisms leading to cell death. The deletion of the NLSI sequence decreases p53 nuclear translocation as well as its mitochondrial localization and consequently its effect on yeast growth.

The assessment of BRCA1 and BRCA2 coding sequences to identify pathogenic mutations associated with inherited breast/ovarian cancer syndrome has provided a method to identify high-risk individuals, allowing them to seek preventative treatments and strategies. However, the current test is expensive, and cannot differentiate between pathogenic variants and those that may be benign. Focusing only on one of the two BRCA partners, we have developed a biological assay for haploinsufficiency of BRCA1. Using a series of EBV-transformed cell lines, we explored gene expression patterns in cells that were BRCA1 wildtype compared to those that carried (heterozygous) BRCA1 pathogenic mutations. We identified a subset of 43 genes whose combined expression pattern is a sensitive predictor of BRCA1 status. The gene set was disproportionately made up of genes involved in cellular differentiation, lending credence to the hypothesis that single copy loss of BRCA1 function may impact differentiation, rendering cells more susceptible to undergoing malignant processes.

The cellular changes during an epithelial-mesenchymal transition (EMT) largely rely on global changes in gene expression orchestrated by transcription factors. Tead transcription factors and their transcriptional co-activators Yap and Taz have been previously implicated in promoting an EMT; however, their direct transcriptional target genes and their functional role during EMT have remained elusive. We have uncovered a previously unanticipated role of the transcription factor Tead2 during EMT. During EMT in mammary gland epithelial cells and breast cancer cells, levels of Tead2 increase in the nucleus of cells, thereby directing a predominant nuclear localization of its co-factors Yap and Taz via the formation of Tead2-Yap-Taz complexes. Genome-wide chromatin immunoprecipitation and next generation sequencing in combination with gene expression profiling revealed the transcriptional targets of Tead2 during EMT. Among these, zyxin contributes to the migratory and invasive phenotype evoked by Tead2. The results demonstrate that Tead transcription factors are crucial regulators of the cellulardistribution of Yap and Taz, and together they control the expression of genes critical for EMT and metastasis.

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a "transformation suppressor" protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G(0)/G(1) phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in beta-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a “transformation suppressor” protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G0/G1 phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in β-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when

Endocrine actions of growth hormone (GH) have been implicated during the development of adult testicular function in several mammalian species, and recently intracrine, autocrine, and paracrine effects have been proposed for locally expressed GH. Previous reports have shown the distribution of GH mRNA and the molecular heterogeneity of GH protein in both adult chicken testes and vas deferens. This study provides evidence of the presence and distribution of GH and its receptor (GHR) during all stages of spermatogenesis in adult chicken testes. This hormone and its receptor are not restricted to the cytoplasm; they are also found in the nuclei of spermatogonia, spermatocytes, and spermatids. The pattern of GH isoforms was characterized in the different, isolated germ cell subpopulations, and the major molecular variant in all subpopulations was 17 kDa GH, as reported in other chicken extra-pituitary tissues. Another molecular variant, the 29 kDa moiety, was found mainly in the enriched spermatocyte population, suggesting that it acts at specific developmental stages. The co-localization of GH with the proliferative cell nuclear antigen PCNA (a DNA replication marker present in spermatogonial cells) was demonstrated by immunohistochemistry. These results show for the first time that GH and GHR are present in the nuclei of adult chicken germinal cells, and suggest that GH could participate in proliferation and differentiation during the complex process of spermatogenesis.

Astacin-like squid metalloprotease (ALSM) is a member of the astacin family of metalloproteases. In the present study, we investigated the expression and tissue distribution of ALSM in bigfin reef squid (Sepioteuthis lessoniana) and golden cuttlefish (Sepia esculenta). Myosin heavy chain hydrolysis tests showed ALSM-I-like activity in both species. We isolated partial cDNA clones showing high sequence similarity to ALSM-I and -III, suggesting that ALSM is common to squid and cuttlefish. Phylogenetic analysis showed that ALSMs are classified into two clades: ALSM-I forms one clade, and ALSM-II and -III form the other. ALSM was expressed in several tissues in bigfin reef squid, though expression was confined to the liver in cuttlefish. ALSMs are distributed in digestive organs but not in mantle muscle of squid and cuttlefish. Immunofluorescence analysis further showed that cellular localization of ALSM is evident not only in hepatic cells but also in pancreatic cells of bigfin reef squid. Thus, ALSM is commonly expressed in squid and cuttlefish, but its expression levels and distribution are distinct.

Retinoids are established pleiotropic regulators of both adaptive and innate immune responses. Recently, troglitazone, a PPAR gamma agonist, has been demonstrated to have anti-inflammatory effects. Separately, retinoids and troglitazone are implicated in immune related processes; however, their combinatory role in cellular adhesion and proliferation has not been well established. In this study, the effect of 9-cis-retinoic acid (9-cis-RA) and troglitazone on K562 cellular adhesion and proliferation was investigated. Troglitazone exposure decreased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin, FN-120, and vitronectin in a concentration and time-dependent manner. In the presence of troglitazone, 9-cis-retinoic acid restores cellular adhesion to levels comparable to vehicle treatment alone on fibronectin, FN-120, and vitronectin substrates within 72 hours. Due to the prominent role of integrins in attachment to extracellular matrix proteins, we evaluated the level of integrin α5 subunit expression. Troglitazone treatment results in decrease in α5 subunit expression on the cell surface. In the presence of both agonists, cell surface α5 subunit expression was restored to levels comparable to vehicle treatment alone. Additionally, troglitazone and 9-cis-RA mediated cell adhesion was decreased in the presence of a function blocking integrin alpha 5 inhibitor. Further, through retinoid metabolic profiling and HPLC analysis, our study demonstrates that troglitazone augments retinoid availability in K562 cells. Finally, we demonstrate that troglitazone and 9-cis-retinoic acid synergistically dampen cellular proliferation in K562 cells. Our study is the first to report that the combination of troglitazone and 9-cis-retinoic acid restores cellular adhesion, alters retinoid availability, impacts integrin expression, and dampens cellular proliferation in K562 cells.

Observation of phenotypic diversity in a population of genetically identical cells is often linked to the stochastic nature of chemical reactions involved in gene regulatory networks. We investigate the distribution of population-averaged gene expression levels as a function of population, or sample, size for several stochastic gene expression models to find out to what extent population-averaged quantities reflect the underlying mechanism of gene expression. We consider three basic gene regulation networks corresponding to transcription with and without gene state switching and translation. Using analytical expressions for the probability generating function of observables and large deviation theory, we calculate the distribution and first two moments of the population-averaged mRNA and protein levels as a function of model parameters, population size, and number of measurements contained in a data set. We validate our results using stochastic simulations also report exact results on the asymptotic properties of population averages which show qualitative differences among different models.

Like severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus (HCoV)-NL63 employs angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry. SARS-CoV infection causes robust downregulation of cellular ACE2 expression levels and it has been suggested that the SARS-CoV effect on ACE2 is involved in the severity of disease. We investigated whether cellular ACE2 downregulation occurs at optimal replication conditions of HCoV-NL63 infection. The expression of the homologue of ACE2, the ACE protein not used as a receptor by HCoV-NL63, was measured as a control. A specific decrease for ACE2 protein level was observed when HCoV-NL63 was cultured at 34 °C. Culturing the virus at the suboptimal temperature of 37 °C resulted in low replication of the virus and the effect on ACE2 expression was lost. We conclude that the decline of ACE2 expression is dependent on the efficiency of HCoV-NL63 replication, and that HCoV-NL63 and SARS-CoV both affect cellular ACE2 expression during infection.

Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., Cereb. Cortex 18, 2789–2795), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of α1-, α2- and β-adrenergic receptors and their subtypes was determined in freshly-isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10–12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. α1A-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and α1B-receptors were not expressed in any cell population. Among α2-receptors only α2A-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. β1-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas β2-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of α2-adrenoceptors

PGE2 has been implicated in abdominal aortic aneurysm (AAA) associated hypervascularization. PGE2-metabolism involves 15-hydroxyprostaglandin-dehydrogenase (15-PGDH) the expression of which in AAA is unknown. The aim of this study was to examine the expression and cell distribution of 15-PGDH in AAA. Here, we show that 15-PGDH mRNA levels were significantly higher in aorta samples from patients undergoing AAA repair than in those from healthy multiorgan donors. Consequently, the ratio of metabolized PGE2 secreted by aortic samples was significantly higher in AAA. AAA production of total PGE2 and PGE2 metabolites correlated positively with PGI2 production, while the percentage of metabolized PGE2 correlated negatively with the total amount of PGE2 and with PGI2. Transcript levels of 15-PGDH were statistically associated with leukocyte markers but did not correlate with microvascular endothelial cell markers. Immunohistochemistry revealed 15-PGDH in the areas of leukocyte infiltration in AAA samples, mainly associated with CD45-positive cells, but not in normal aorta samples. We provide new data concerning 15-PGDH expression in human AAA, showing that 15-PGDH is upregulated in AAA and mainly expressed in infiltrating leukocytes. Our data suggest that microvasculature was not involved in PGE2 catabolism, reinforcing the potential role of microvasculature derived PGE2 in AAA-associated hypervascularization. PMID:26287481

The present study assessed the influence of salinity and temperature on body growth and on muscle cellularity of Lophiosilurus alexaxdri vitelinic larvae. Slightly salted environments negatively influenced body growth of freshwater fish larvae and we observed that those conditions notably act as an environmental influencer on muscle growth and on local expression of hypertrophia and hypeplasia markers (IGFs and PCNA). Furthermore, we could see that salinity tolerance for NaCl 4gl(-)(1) diminishes with increasing temperature, evidenced by variation in body and muscle growth, and by irregular morphology of the lateral skeletal muscle of larvae. We saw that an increase of both PCNA and autocrine IGF-II are correlated to an increase in fibre numbers and fibre diameter as the temperature increases and salinity diminishes. On the other hand, autocrine IGF-I follows the opposite way to the other biological parameters assessed, increasing as salinity increases and temperature diminishes, showing that this protein did not participate in muscle cellularity, but participating in molecular/cellular repair. Therefore, slightly salted environments may provide adverse conditions that cause some obstacles to somatic growth of this species, suggesting some osmotic expenditure with a salinity increment.

On the basis of its CO(2) compensation concentration, Brassica gravinae Ten. has been reported to be a C(3)-C(4) intermediate. This study investigated the structural and biochemical features of photosynthetic metabolism in B. gravinae. The cellulardistribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was also examined in B. gravinae, B. napus L. (C(3)), Raphanus sativus L. (C(3)), and Diplotaxis tenuifolia (L.) DC. (C(3)-C(4)) by immunogold electron microscopy to elucidate Rubisco expression during the evolution from C(3) to C(3)-C(4) intermediate plants. The bundle sheath (BS) cells of B. gravinae contained centrifugally located chloroplasts as well as centripetally located chloroplasts and mitochondria. Glycine decarboxylase P-protein was localized in the BS mitochondria. Brassica gravinae had low C(4) enzyme activities and high activities of Rubisco and photorespiratory enzymes, suggesting that it reduces photorespiratory CO(2) loss by the glycine shuttle. In B. gravinae, the labelling density of Rubisco was higher in the mesophyll chloroplasts than in the BS chloroplasts. A similar cellular pattern was found in other Brassicaceae species. These data demonstrate that, during the evolution from C(3) to C(3)-C(4) intermediate plants, the intercellular pattern of Rubisco expression did not change greatly, although the amount of chloroplasts in the BS cells increased. It also appears that intracellular variation in Rubisco distribution may occur within the BS cells of B. gravinae.

A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.

It has been reported that Dictyostelium discoideum encodes four silent information regulator 2 (Sir2) proteins (Sir2A-D) showing sequence similarity to human homologues of Sir2 (SIRT1-3). Further screening in a database revealed that D. discoideum encodes an additional Sir2 homologue (Sir2E). The amino acid sequence of Sir2E is not similar to those of SIRTs but is similar to those of proteins encoded by Giardia lamblia, Cryptosporidium hominis and Cryptosporidium parvum. Fluorescence of Sir2E-green fluorescent protein fusion protein was detected in the D. discoideum nucleus, indicating that Sir2E is a nuclear localizing protein. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that D. discoideum expressed sir2E in amoebae in the growth phase and in prestalk cells in the developmental phase. D. discoideum overexpressing sir2E grew faster than the wild type. These results indicate that Sir2E plays important roles both in the growth phase and developmental phase of D. discoideum.

Background While the transcription of innate immunity genes in response to bacterial infection has been well-characterised in the Drosophila model, we recently demonstrated the capacity for such transcription to evolve in flies selected for improved antibacterial defense. Here we use this experimental system to examine how insects invest in constitutive versus infection-induced transcription of immunity genes. These two strategies carry with them different consequences with respect to energetic and pleiotropic costs and may be more or less effective in improving defense depending on whether the genes contribute to humoral or cellular aspects of immunity. Findings Contrary to expectation we show that selection preferentially increased the infection-induced expression of both cellular and humoral immunity genes. Given their functional roles, infection induced increases in expression were expected for the humoral genes, while increases in constitutive expression were expected for the cellular genes. We also report a restricted ability to improve transcription of immunity genes that is on the order of 2-3 fold regardless of total transcription level of the gene. Conclusions The evolved increases in infection-induced expression of the cellular genes may result from specific cross talk with humoral pathways or from generalised strategies for enhancing immunity gene transcription. A failure to see improvements in constitutive expression of the cellular genes suggests either that increases might come at too great a cost or that patterns of expression in adults are decoupled from the larval phase where increases would be most effective. The similarity in fold change increase across all immunity genes may suggest a shared mechanism for the evolution of increased transcription in small, discrete units such as duplication of cis-regulatory elements. PMID:21859495

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates splice site usage, polyadenylation, and cleavage efficiency. This protein has also been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that hnRNP A1 had diminished protein levels and showed cytoplasmic accumulation in senescent human diploid fibroblasts. Furthermore, we have shown that inhibition of p38 MAPK, a key regulator of cellular senescence, elevated hnRNP A1 protein levels and inhibited hnRNP A1 cytoplasmic localization. In this study, we have explored the possible involvement of MNK1, one of the downstream effector of p38 MAPK, in the regulation of hnRNP A1. We have demonstrated that pharmacological inhibition of MNK1 by CGP 57380 decreased the phosphorylation levels of hnRNP A1 in young and senescent fibroblast cells and blocked the cytoplasmic accumulation of hnRNP A1 in senescent cells. In addition, MNK1 formed a complex with hnRNP A1 in vivo. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins were found to be elevated in senescent cells. These data suggest that MNK1 regulates the phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the induction of senescence. -- Highlights: Black-Right-Pointing-Pointer MNK1 and not MAPKAPK2 phosphorylates hnRNP A1. Black-Right-Pointing-Pointer MNK1 has elevated levels in senescent cells, this has not been reported previously. Black-Right-Pointing-Pointer MNK1 activity induces cytoplasmic accumulation of hnRNP A1 in senescent cells. Black-Right-Pointing-Pointer Altered cytoplasmic localization of hnRNP A1 may alter gene expression patterns. Black-Right-Pointing-Pointer Our studies may increase our understanding of RNA metabolism during cellular aging.

Background To model and thoroughly understand animal transcription networks, it is essential to derive accurate spatial and temporal descriptions of developing gene expression patterns with cellular resolution. Results Here we describe a suite of methods that provide the first quantitative three-dimensional description of gene expression and morphology at cellular resolution in whole embryos. A database containing information derived from 1,282 embryos is released that describes the mRNA expression of 22 genes at multiple time points in the Drosophila blastoderm. We demonstrate that our methods are sufficiently accurate to detect previously undescribed features of morphology and gene expression. The cellular blastoderm is shown to have an intricate morphology of nuclear density patterns and apical/basal displacements that correlate with later well-known morphological features. Pair rule gene expression stripes, generally considered to specify patterning only along the anterior/posterior body axis, are shown to have complex changes in stripe location, stripe curvature, and expression level along the dorsal/ventral axis. Pair rule genes are also found to not always maintain the same register to each other. Conclusion The application of these quantitative methods to other developmental systems will likely reveal many other previously unknown features and provide a more rigorous understanding of developmental regulatory networks. PMID:17184546

The β1 nicotinic acetylcholine receptor (nAChR) subunit is a muscle type subunit of this family and as such, is found predominantly in muscle. Recent reports document its expression in other tissues and cell lines including adrenal glands, carcinomas, lung and brain. However, the majority of studies were of tissue lysates, thus the cellulardistribution was not determined. This study aimed to determine the cellulardistribution of the β1 nAChR subunit in the brain, at both the mRNA and protein levels, using non-radioactive in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, and to compare it to two muscle tissue types, skeletal and placenta. Tissue was formalin fixed and paraffin embedded (all tissue types) and frozen (placenta) from humans. Additional control tissue from the piglet and mouse brain were also studied, as was mRNA for the α3 nAChR and N-methyl-d-aspartate receptor 1 (NR1) subunit. We found no β1 nAChR subunit mRNA expression in the human and piglet brain despite strong protein expression. Some signal was seen in the mouse brain but considered inconclusive given the probes designed were not of 100% homology to the mouse. In the skeletal muscle and placenta tissues, β1 nAChR subunit mRNA expression was prominent and mirrored protein expression. No α3 nAChR or NR1 mRNA was seen in the skeletal muscle, as expected, although both subunit mRNAs were present in the placenta. This study concludes that further experiments are required to conclusively state that the β1 nAChR subunit is expressed in the human, piglet and mouse brain.

Stochasticity in gene expression can give rise to fluctuations in protein levels and lead to phenotypic variation across a population of genetically identical cells. Recent experiments indicate that bursting and feedback mechanisms play important roles in controlling noise in gene expression and phenotypic variation. A quantitative understanding of the impact of these factors requires analysis of the corresponding stochastic models. However, for stochastic models of gene expression with feedback and bursting, exact analytical results for protein distributions have not been obtained so far. Here, we analyze a model of gene expression with bursting and feedback regulation and obtain exact results for the corresponding protein steady-state distribution. The results obtained provide new insights into the role of bursting and feedback in noise regulation and optimization. Furthermore, for a specific choice of parameters, the system studied maps on to a two-state biochemical switch driven by a bursty input noise source. The analytical results derived provide quantitative insights into diverse cellular processes involving noise in gene expression and biochemical switching.

SKP1 is a core component of SCF complex, a major type of E3 ubiquitin ligase catalyzing the last step in ubiquitin-mediated protein degradation pathway. In present study, SKP1 gene family in Solanum pimpinellifolium (SSK), a wild species of tomato, was investigated. A total of 19 SSK genes were identified through homologous search. Their chromosomal locations, gene structures, phylogeny, expression profiles, sub-cellular localizations and protein-protein interaction patterns with putative F-box proteins were analyzed in detail. The high homology and similar expression patterns among clustered SSK genes in chromosome suggested that they may have evolved from duplication events and are functionally redundant. Sub-cellular localization indicated that most of the SSK proteins are distributed in both cytosol and nucleus, except for SSK8, which is detected in cytosol only. Tissue-specific expression patterns suggested that many SSK genes may be involved in tomato fruit development. Furthermore, several SSK genes were found to be responsive to heat stress and salicylic acid treatment. Based on phylogenetic analysis, expression profiles and protein interaction property, we proposed that tomato SSK1 and SSK2 might have similar function to ASK1 and ASK2 in Arabidopsis.

Ferritin is a ubiquitously distributed iron-binding protein. Cell culture studies have demonstrated that ferritin plays a role in maintenance of iron homoeostasis and in the protection against cytokine- and oxidant-induced stress. To test whether FerH (ferritin H) can regulate tissue iron homoeostasis in vivo, we prepared transgenic mice that conditionally express FerH and EGFP (enhanced green fluorescent protein) from a bicistronic tetracycline-inducible promoter. Two transgenic models were explored. In the first, the FerH and EGFP transgenes were controlled by the tTACMV (Tet-OFF) (where tTA and CMV are tet transactivator protein and cytomegalovirus respectively). In skeletal muscle of mice bearing the FerH/EGFP and tTACMV transgenes, FerH expression was increased 6.0±1.1-fold (mean±S.D.) compared with controls. In the second model, the FerH/EGFP transgenes were controlled by an optimized Tet-ON transactivator, rtTA2S-S2LAP (where rtTA is reverse tTA and LAP is liver activator protein), resulting in expression predominantly in the kidney and liver. In mice expressing these transgenes, doxycycline induced FerH in the kidney by 14.2±4.8-fold (mean±S.D.). Notably, increases in ferritin in overexpressers versus control littermates were accompanied by an elevation of IRP (iron regulatory protein) activity of 2.3±0.9-fold (mean±S.D.), concurrent with a 4.5±2.1-fold (mean±S.D.) increase in transferrin receptor, indicating that overexpression of FerH is sufficient to elicit a phenotype of iron depletion. These results demonstrate that FerH not only responds to changes in tissue iron (its classic role), but can actively regulate overall tissue iron balance. PMID:16448386

A massive expansion of a GGGGCC repeat upstream of the C9orf72 coding region is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. Despite its intronic localization and lack of a canonical start codon, both strands are translated into aggregating dipeptide repeat (DPR) proteins: poly-GA, poly-GP, poly-GR, poly-PR and poly-PA. To address conflicting findings on the predominant toxicity of the different DPR species in model systems, we compared the expression pattern of the DPR proteins in rat primary neurons and postmortem brain and spinal cord of C9orf72 mutation patients. Only poly-GA overexpression closely mimicked the p62-positive neuronal cytoplasmic inclusions commonly observed for all DPR proteins in patients. In contrast, overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions. In patients, most of the less common neuronal intranuclear DPR inclusions were para-nucleolar and p62 positive. Neuronal nucleoli in C9orf72 cases showed normal size and morphology regardless of the presence of poly-GR and poly-PR inclusions arguing against widespread nucleolar stress, reported in cellular models. Colocalization of para-nucleolar DPR inclusions with heterochromatin and a marker of transcriptional repression (H3K9me2) indicates a link to gene transcription. In contrast, we detected numerous intranuclear DPR inclusions not associated with nucleolar structures in ependymal and subependymal cells. In patients, neuronal inclusions of poly-GR, poly-GP and the poly-GA interacting protein Unc119 were less abundant than poly-GA inclusions, but showed similar regional and subcellular distribution. Regardless of neurodegeneration, all inclusions were most abundant in neocortex, hippocampus and thalamus, with few inclusions in brain stem and spinal cord. In the granular cell layer of the cerebellum, poly-GA and Unc119 inclusions were significantly more abundant in cases with FTLD than in cases with MND and FTLD/MND. Poly

Galectin is an animal lectin that recognizes beta-galactosides of glycoconjugates and is abundant in the gut. This study revealed the cellularexpression of galectin subtypes throughout the mouse digestive tract by in situ hybridization. Signals for five subtypes (galectin-2, -3, -4/6, and -7) were detected exclusively in the epithelia. In the glandular stomach, galectin-2 and -4/6 were predominantly expressed from gastric pits to neck of gastric glands, where mucous cells were the main cellular sources. The small intestine exhibited intense, maturation-associated expressions of galectin-2, -3, and -4/6 mRNAs. Galectin-2 was intensely expressed from crypts to the base of villi, whereas transcripts of galectin-3 gathered at villous tips. Signals for galectin-4/6 were most intense at the lower half of villi. Galectin-2 was also expressed in goblet cells of the small intestine but not in those of the large intestine. In the large intestine, galectin-4/6 predominated, and the upper half of crypts simultaneously contained transcripts of galectin-3. Stratified epithelium from the lip to forestomach and anus intensely expressed galectin-7 with weak expressions of galectin-3. Because galectins in the digestive tract may be multi-functional, information on their cell/stage-specific expression contributes to a better understanding of the functions and pathological involvements of galectins.

Recent studies have investigated the expression of proliferative markers, but little is known about the expression of cytokeratin 15 and 19 in different histological subtypes of basal cell carcinoma (BCC). We conducted cellular, biological, clinicopathological and immunohistochemical analysis on the manifestations of 8 BCC by hematoxylin and eosin stain (H&E) staining and immunohistochemistry and reviewed relevant literature. Microscopically, the tumor cells were multiple remarkable foci of epidermolytic hyperkeratosis with large pleomorphic nuclei and scant cytoplasm together with peripheral palisading and forming solid nests. Furthermore, the most tumors were composed of highly cellular areas with a homogenous population of round, ovoid and spindle cells, hyperchromatic nuclei, high cellular pleomorphism, high mitotic index and various morphologic patterns. Moreover, the tumors displayed an invasive growth, with positive expression of Cytokeratin 19 (CK19) and negative expression of CK15. Our study revealed that the expression of CK19 was associated with progression and invasion in cases with BCC and immunohistochemistry is indispensable in distinguishing this tumor from other types of cutaneous carcinoma. To our best knowledge, it may be a considerable biomarker to assess invasiveness of cutaneous-surface BCC and to guide clinical management of such tumors.

Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation. PMID:27478062

Endocan is a novel human endothelial cell specific molecule. Its expression is regulated by cytokines and vascular endothelial growth factor (VEGF). The distribution of endocan in normal human tissues, however, remains unclear. We examined the expression of endocan in normal human tissue using immunohistochemical stains. Endocan was expressed in actively proliferative or neogeneic tissues and cells such as glandular tissues, endothelium of neovasculature, bronchial epithelium, germinal centers of lymph nodes etc. Endocan was not present in silent or resting tissues or cells such as endothelium of great arteries and spleen etc. Our findings suggest that endocan may act as a marker for angiogenesis or oncogenesis and could be regarded as a candidate gene for inflammatory tissue, neoplasia, tumor development and metastasis. The expression level of endocan may assist early diagnosis and prognosis of some tumors.

Tissue transglutaminase is a cytosolic enzyme whose primary function is to catalyze the covalent cross-linking of proteins. To investigate the functions of this enzyme in physiological systems, we have established lines of Balb-C 3T3 fibroblasts stably transfected with a constitutive tissue transglutaminase expression plasmid. Several cell lines expressing high levels of catalytically active tissue transglutaminase have been isolated and characterized. Transglutaminase-transfected cells showed morphologic features quite distinct from their nontransfected counterparts. Many of the cells showed an extended and very flattened morphology that reflected increased adhesion of the cells to the substratum. Other cells, particularly those showing the highest levels of intracellular transglutaminase expression, showed extensive membrane blebbing and cellular fragmentation. The results of these experiments suggest that the induction and activation of tissue transglutaminase may contribute both to changes in cellular morphology and adhesiveness. PMID:1356992

The microscope and infrared spectrometer are two of the most useful tools for the study of biological materials, and their combined analytical power far exceeds the sum of the two. Performing molecular spectroscopy through a microscope superimposes chemical information onto the physical microstructure obtained from the optical microscope when visible and infrared information are collected under the same conditions. The instrument developments that enable current infrared microspectroscopic studies began with the introduction of the first research-grade infrared microscope, patented in 1989 (1). By 1993, published reports using this method to determine macroalgae (seaweed) cell-wall composition appeared (2-4). Since these initial reports, the use of infrared microspectroscopy (IMS) in microalgal (single cells or groups of cells) research has grown. Primarily, cultured algae have been used to hone IMS methodology and evaluate its capabilities in algal research (5-8). Studies involving natural, mixed species assemblages, which can utilize the spatial resolution potential of this technique fully are rare (9-11). For instance, in a recent review of IMS microalgal ecological research (12), only 3 of the 29 peer-reviewed publications investigated natural algal assemblages. Both thermal and synchrotron infrared sources provide a resolution capable of measuring individual algae in mixed species assemblages, and each has its advantages. For example, thermal source IMS is more accessible, allowing more samples to be analyzed than synchrotron IMS. However, synchrotron IMS with confocal masking provides superior resolution, which can be critical in isolating small or contiguous cells. Algal ecology is the study of the interaction between algae and their environment. Infrared microspectroscopy addresses a major logistical problem in this field, obtaining species-specific cellular biochemical information from natural, mixed-species assemblages (11,12). Benthic (bottom

Observation of phenotypic diversity in a population of genetically identical cells is often linked to the stochastic nature of chemical reactions involved in gene regulatory networks. We investigate the distribution of population averaged gene expression levels as a function of population, or sample size for several stochastic gene expression models to find out to what extent population averaged quantities reflect the underlying mechanism of gene expression. We consider three basic gene regulation networks corresponding to transcription with and without gene state switching and translation. Using analytical expressions for the probability generating function (pgf) of observables and Large Deviation Theory, we calculate the distribution of population averaged mRNA and protein levels as a function of model parameters and population size. We validate our results using stochastic simulations also report exact results on the asymptotic properties of population averages which show qualitative differences for different models. We calculate the skewness and coefficient of variance for pgfs to estimate the sample size required for population average that contains information about gene expression models. This is relevant to experiments where a large number of data points are unavailable.

Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1-296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellulardistribution of sHSPs.

Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1–296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellulardistribution of sHSPs. PMID:26359114

The distribution of leucocyte common antigen (LCA) protein subspecies and the cellular adhesion molecules LFA-1 (CD11a), ICAM-1 (CD54) and p150,95 (CD11c) has been established within frozen sections of human foetal thymus. Whereas over 95% of foetal cortical thymocytes and approximately 85% of medullary thymocytes were CD45RO positive, CD45RA was only expressed by approximately 29% of medullary thymocytes. The majority of foetal thymocytes also expressed CD11a, whereas CD54 was expressed by thymic epithelial and accessory cells and also apparently by some cortical thymocytes adjacent to epithelial cells. The distribution of CD54 and the major histocompatibility complex (MHC) class II molecule HLA-DR, demonstrated with a monoclonal antibody to a monomorphic determinant, was similar. The CD11c molecule was present on a population of dendritic-type accessory cells, but was absent from the large, scavenger, KiM8-positive macrophages occurring throughout the thymic cortex. Images Figure 1 Figure 2 PMID:1973681

Cancer cells treated with the cyclooxygenase-2 inhibitor celecoxib show growth inhibition and induced apoptosis. This study was conducted to determine if the same processes are relevant to celecoxib's effects on human colorectal adenocarcinomas treated in vivo. A cohort of 23 patients with primary colorectal adenocarcinomas was randomised to receive a 7-d course of celecoxib (400mg b.i.d.) or no drug prior to surgical resection. Gene expression profiling was performed on resected adenocarcinomas from the cohort of patients. Using fold change (>1.5) and p-value (<0.05) cut-offs, 190 genes were differentially expressed between adenocarcinomas from patients receiving celecoxib and those that did not. The celecoxib pre-treated samples showed decreased expression levels in multiple genes involved in cellular lipid and glutathione metabolism; changes associated with diminished cellular proliferation. Celecoxib pre-treatment for 7 d in vivo is associated with alterations in colorectal adenocarcinoma gene expression which are suggestive of diminished cellular proliferation.

Cancer cells treated with the cyclooxygenase-2 inhibitor celecoxib show growth inhibition and induced apoptosis. This study was conducted to determine if the same processes are relevant to celecoxib’s effects on human colorectal adenocarcinomas treated in vivo. A cohort of 23 patients with primary colorectal adenocarcinomas was randomized to receive a 7-day course of celecoxib (400 mg b.i.d.) or no drug prior to surgical resection. Gene expression profiling was performed on resected adenocarcinomas from the cohort of patients. Using fold change (>1.5) and p-value (<0.05) cut-offs, 190 genes were differentially expressed between adenocarcinomas from patients receiving celecoxib and those that did not. The celecoxib pre-treated samples showed decreased expression levels in multiple genes involved in cellular lipid and glutathione metabolism; changes associated with diminished cellular proliferation. Celecoxib pre-treatment for 7 days in vivo is associated with alterations in colorectal adenocarcinoma gene expression which are suggestive of diminished cellular proliferation. PMID:18653328

Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of γ-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer. The cell cycle was arrested in the G(0)/G(1) phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G(0)/G(1) phase and increased cell populations in the G(2)/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts. γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.

Preadipocyte factor-1 (pref-1) is specifically expressed in preadipocytes and acts as a gatekeeper of adipogenesis by maintaining the preadipocyte state and preventing adipocyte differentiation. We hypothesized that the breed differences of adipogenic capacity in cattle could be explained by the expression level of pref-1. In this experiment, we studied the expression level of the pref-1 gene and adipocyte cellularity in subcutaneous and mesenteric adipose tissues of Japanese Black (Wagyu) and Holstein fattening cattle. In subcutaneous adipose tissue, there were no significant differences in the pref-1 gene expression levels and adipocyte sizes between the breeds. In contrast, the expression level of the pref-1 gene in mesenteric adipose tissue of Holsteins was significantly higher than that of Wagyu. In addition, the size of mesenteric adipocytes in Holsteins was significantly smaller than that of Wagyu. These results indicate that the breed differences of fattening cattle affect the expression pattern of the pref-1 gene and adipocyte cellularity in a fat depot-specific manner.

Living organisms in space are constantly exposed to radiation, toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage is essential for assessing the radiation risk for astronauts and the mutation rate in microorganisms. In a study conducted on the International Space Station, confluent human fibroblasts in culture were treated with bleomycin for three hours in the true microgravity environment. The degree of DNA damage was quantified by immunofluorescence staining for γ-H2AX, which is manifested in three types of staining patterns. Although similar percentages of these types of patterns were found between flight and ground cells, there was a slight shift in the distribution of foci counts in the flown cells with countable numbers of γ-H2AX foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. We also performed a microarray analysis of gene expressions in response to bleomycin treatment. A qualitative comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. The microarray data was confirmed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly upregulated in both flight and ground cells after bleomycin treatment. Our results suggest that whether microgravity affects DNA damage response in space can be dependent on the cell type and cell growth condition. PMID:28248986

Insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1), a member of the IGFBP super family, was identified as a potent tumor suppressor in several carcinomas. IGFBP-rP1 was down-regulated in primary breast cancer tissues and several breast cancer cell lines but overexpressed in senescent human mammary epithelial cells (HMECs), suggesting that IGFBP-rP1 might be a tumor suppressor in breast cancer and the tumor suppressor role of IGFBP-rP1 might be associated with cellular senescence. The aim of the study was to observe the effect of IGFBP-rP1 on cellular senescence and the molecular events mediating this biological effect in MCF-7 breast cancer cells. DNA fragment-encoding IGFBP-rP1 was cloned in-frame N-terminally to EGFP gene to generate IGFBP-rP1-EGFP fusion protein expression plasmid (pEGFP-IGFBP-rP1). The plasmid pEGFP-IGFBP-rP1 was then transfected into MCF-7 cells, and the proliferation, cell cycle distribution, cellular senescence, and cell cycle-related protein expression of MCF-7 cells were examined by trypan blue exclusion, flow cytometry, senescence-associated galactosidase (SA-β-gal) staining, and Western blot analysis, respectively. Two shRNA plasmid vectors against p21 or p53 gene were constructed and stably transfected into the MCF-7 cells to determine the involvement of p21 or p53 in cellular senescence induced by IGFBP-rP1. Transfection of IGFBP-rP1 or addition of condition medium (CM) from IGFBP-rP1-transfected cells in MCF-7 cells caused induction of a variety of senescent phenotypes, such as decrease in cell proliferation, increase in G0/G1 cell cycle arrest cells, change in cell morphology, and increase in senescence-associated galactosidase (SA-β-gal) activity. IGFBP-rP1-induced growth arrest is associated with enhanced expression of the cyclin-dependent kinase inhibitor p21 and dephosphorylation of the retinoblastoma protein (pRB). Cell proliferation block and cellular senescence induction in response to IGFBP-rP1

Modified melanoma B16 cells inhibited in their IGF-1 expression (B16MOD), on the contrary to the IGF-1 fully expressed parental wild-type (B16WT) counterpart, were shown to stimulate humoral as well as cellular immune responses. Among humoral components, the neutralizing and complement-fixing antibodies of IgM and essentially IgG2 (a+b) isotypes exhibited in vitro and in vivo effects upon tumour growth, while the IgG1 antibody isotype promoted enhanced tumour proliferation. As for the cellular immunity, it was found that the T CD8(+) lymphocyte subpopulation remained the main potent and long lasting immune active effector regulating tumour growth.

Poorly differentiated tumors in non-small cell lung cancer (NSCLC) have been associated with shorter patient survival and shorter time to recurrence following treatment. Here, we integrate multiple experimental models with clinicopathologic analysis of patient tumors to delineate a cellular hierarchy in NSCLC. We show that the oncofetal protein 5T4 is expressed on tumor-initiating cells and associated with worse clinical outcome in NSCLC. Coexpression of 5T4 and factors involved in the epithelial-to-mesenchymal transition were observed in undifferentiated but not in differentiated tumor cells. Despite heterogeneous expression of 5T4 in NSCLC patient-derived xenografts, treatment with an anti-5T4 antibody-drug conjugate resulted in complete and sustained tumor regression. Thus, the aggressive growth of heterogeneous solid tumors can be blocked by therapeutic agents that target a subpopulation of cells near the top of the cellular hierarchy.

Mammalian oocyte growth and development is driven by a strict program of gene expression that relies on the timely presence of transcriptional regulators via nuclear pores. By targeting specific cargos for nucleo-cytoplasmic transport, karyopherin (KPN) proteins are key to the relocation of essential transcription factors and chromatin-remodelling factors into and out of the nucleus. Using multiple complementary techniques, here we establish that KPNA genes and proteins are dynamically expressed and relocalised throughout mouse oogenesis and folliculogenesis. Of the KPNAs examined (Kpna1, Kpna2, Kpna3, Kpna4, Kpna6, Kpna7, Kpnb1, Ipo5 and Xpo1), all were expressed in the embryonic ovary with up-regulation of protein levels concomitant with meiotic entry for KPNA2, accompanied by the redistribution of the cellular localisation of KPNA2 and XPO1. In contrast, postnatal folliculogenesis revealed significant up-regulation of Kpna1, Kpna2, Kpna4, Kpna6 and Ipo5 and down-regulation of Kpnb1, Kpna7 and Xpo1 at the primordial to primary follicle transition. KPNAs exhibited different localisation patterns in both oocytes and granulosa cells during folliculogenesis, with three KPNAs--KPNA1, KPNA2 and IPO5--displaying marked enrichment in the nucleus by antral follicle stage. Remarkably, varied subcellular expression profiles were also identified in isolated pre-ovulatory oocytes with KPNAs KPNA2, KPNB1 and IPO5 detected in the cytoplasm and at the nuclear rim and XPO1 in cytoplasmic aggregates. Intriguingly, meiotic spindle staining was also observed for KPNB1 and XPO1 in meiosis II eggs, implying roles for KPNAs outside of nucleo-cytoplasmic transport. Thus, we propose that KPNAs, by targeting specific cargoes, are likely to be key regulators of oocyte development.

Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16, and unique gene expression profile. So far, the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study, the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers, and proteins involved in immune regulation. Within the three human monocyte subsets, our descriptive findings show multimodal expression of key immune response genes, such as CD40, NFⱪB1, RELA, TLR4, TLR8 and TLR9. Furthermore, we discovered one subgroup of cells within the classical monocytes, which showed alterations of 22 genes e.g. IRF8, CD40, CSF1R, NFⱪB1, RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes, respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation, independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance, and can be useful in unravelling inter-cellular variation in leukocyte populations, identifying subpopulations involved in disease pathogenesis and help tailor new therapies. PMID:26650546

Aquaporins (AQPs) are small integral membrane proteins with 13 members in mammals and are essential for water transport across membranes. They are found in many different tissues and cells. Currently, there are conflicting results regarding retinal aquaporin expression and subcellular localization between genome and protein analyses and among various species. AQP4, 7, 9 and 11 were described in the retina of men; whereas AQP6, 8 and 10 were earlier identified in rat retinas and AQP4, 5 and 11 in horses. Since there is a lack of knowledge regarding AQP expression on protein level in retinas of different animal models, we decided to analyze retinal cellularexpression of AQP4, 5 and 11 in situ with immunohistochemistry. AQP4 was detected in all 15 explored species, AQP5 and AQP11 in 14 out of 15. Interestingly, AQP4 was unambiguously expressed in Muller glial cells, whereas AQP5 was differentially allocated among the species analyzed. AQP11 expression was Muller glial cell-specific in 50% of the animals, whereas in the others, AQP11 was detected in ganglion cell layer and at photoreceptor outer segments. Our data indicate a disparity in aquaporin distribution in retinas of various animals, especially for AQP5 and 11.

Aquaporins (AQPs) are small integral membrane proteins with 13 members in mammals and are essential for water transport across membranes. They are found in many different tissues and cells. Currently, there are conflicting results regarding retinal aquaporin expression and subcellular localization between genome and protein analyses and among various species. AQP4, 7, 9 and 11 were described in the retina of men; whereas AQP6, 8 and 10 were earlier identified in rat retinas and AQP4, 5 and 11 in horses. Since there is a lack of knowledge regarding AQP expression on protein level in retinas of different animal models, we decided to analyze retinal cellularexpression of AQP4, 5 and 11 in situ with immunohistochemistry. AQP4 was detected in all 15 explored species, AQP5 and AQP11 in 14 out of 15. Interestingly, AQP4 was unambiguously expressed in Muller glial cells, whereas AQP5 was differentially allocated among the species analyzed. AQP11 expression was Muller glial cell-specific in 50% of the animals, whereas in the others, AQP11 was detected in ganglion cell layer and at photoreceptor outer segments. Our data indicate a disparity in aquaporin distribution in retinas of various animals, especially for AQP5 and 11. PMID:27438827

This paper is concerned with the delay-dependent exponential passivity analysis issue for uncertain cellular neural networks with discrete and distributed time-varying delays. By decomposing the delay interval into multiple equidistant subintervals and multiple nonuniform subintervals, a suitable augmented Lyapunov-Krasovskii functionals are constructed on these intervals. A set of novel sufficient conditions are obtained to guarantee the exponential passivity analysis issue for the considered system. Finally, two numerical examples are provided to demonstrate the effectiveness of the proposed results.

A new method for the qualitative and quantitative determination of the molecular weight distribution of total cellular fatty acids is described. The method includes a simple extraction-saponification followed by chemical ionization-mass spectrometric analysis of the saponifiable matter. This technique requires small quantities of cell material which, combined with the rapidity and precision of the analysis, makes it attractive to the biologist interested in changes in the fatty acid composition of growing cells. As an example, an application of this method to the fatty acid determination of marine diatoms at different growth stages is presented.

Objectives: In about 10% of glioblastoma patients, preoperative MRI discloses the presence of tumor cysts. Whereas the impact of cystic appearance on prognosis has been discussed extensively, only little is known about the tumor cyst fluid. In this study, we tested the feasibility of the surface enhanced laser desorption ionization time of flight (SELDI-TOF) technique to detect cyst fluid proteins. Methods: Cyst fluid was collected from 21 glioblastoma patients for SELDI-TOF analysis and compared to control cerebrospinal fluids from 15 patients with spinal stenosis. Resulting protein peaks with significant differences between groups were further described, using the molecular weight in an internet search of protein databases and publications. Two potential cyst fluid proteins, basigin and ferritin light chain, were selected for immunohistological detection in the histologic slides of the patients, metallothionein (MT) served as negative control. Results: As supposed from the results of the SELDI-TOF analysis, basigin and ferritin were detected immunohistochemically in the cyst wall, whereas MT was more equally distributed between the cyst wall and the surrounding tumor tissue. Median survival time of the patients was 20 months (range 2 to 102 months) and correlated with age, but not with expression of the three proteins. Discussion: The SELDI-TOF approach reveals a number of proteins, potentially present in glioblastoma cyst fluid. Identification of these proteins in tumor cells may help understand the pathogenetic pathways and the prognostic value of cystic changes. PMID:24225180

A critical role of circadian oscillators in orchestrating insulin secretion and islet gene transcription has been demonstrated recently. However, these studies focused on whole islets and did not explore the interplay between α-cell and β-cell clocks. We performed a parallel analysis of the molecular properties of α-cell and β-cell oscillators using a mouse model expressing three reporter genes: one labeling α cells, one specific for β cells, and a third monitoring circadian gene expression. Thus, phase entrainment properties, gene expression, and functional outputs of the α-cell and β-cell clockworks could be assessed in vivo and in vitro at the population and single-cell level. These experiments showed that α-cellular and β-cellular clocks are oscillating with distinct phases in vivo and in vitro. Diurnal transcriptome analysis in separated α and β cells revealed that a high number of genes with key roles in islet physiology, including regulators of glucose sensing and hormone secretion, are differentially expressed in these cell types. Moreover, temporal insulin and glucagon secretion exhibited distinct oscillatory profiles both in vivo and in vitro. Altogether, our data indicate that differential entrainment characteristics of circadian α-cell and β-cell clocks are an important feature in the temporal coordination of endocrine function and gene expression. PMID:28275001

A critical role of circadian oscillators in orchestrating insulin secretion and islet gene transcription has been demonstrated recently. However, these studies focused on whole islets and did not explore the interplay between α-cell and β-cell clocks. We performed a parallel analysis of the molecular properties of α-cell and β-cell oscillators using a mouse model expressing three reporter genes: one labeling α cells, one specific for β cells, and a third monitoring circadian gene expression. Thus, phase entrainment properties, gene expression, and functional outputs of the α-cell and β-cell clockworks could be assessed in vivo and in vitro at the population and single-cell level. These experiments showed that α-cellular and β-cellular clocks are oscillating with distinct phases in vivo and in vitro. Diurnal transcriptome analysis in separated α and β cells revealed that a high number of genes with key roles in islet physiology, including regulators of glucose sensing and hormone secretion, are differentially expressed in these cell types. Moreover, temporal insulin and glucagon secretion exhibited distinct oscillatory profiles both in vivo and in vitro. Altogether, our data indicate that differential entrainment characteristics of circadian α-cell and β-cell clocks are an important feature in the temporal coordination of endocrine function and gene expression.

Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

The cellular location of N-acetylgalactosamine in Bacillus subtilis strains 168 and 170 was examined by electron microscopy using gold-conjugated soybean agglutinin (SBA) as a marker. Post-embedding labeling of sectioned material showed SBA-reactive, galactosamine-containing polymers associated with the cell membrane or the cytoplasm of the two strains. This intracellular location was distinct from the concanavalin A-binding epitopes that were located over the cell wall. Labeling of whole cells (native, fixed in glutaraldehyde, or treated with proteinase K, or Tween 20) before negative staining revealed no galactosamine exposed on the surface of strain 168. On the surface of strain 168 some exposed galactosamine terminal residues were detected; their accessibility to SBA increased when Tween 20 or proteinase K was applied.

When presented with noxious stimuli, "Aplysia" exhibits concurrent sensitization of defensive responses, such as the tail-induced siphon withdrawal reflex (TSWR) and suppression of feeding. At the cellular level, sensitization of the TSWR is accompanied by an increase in the excitability of the tail sensory neurons (TSNs) that elicit the…

The race to manage the health concerns related to excess fat deposition has spawned a proliferation of clinical and basic research efforts to understand variables including dietary uptake, metabolism, and lipid deposition by adipocytes. A full appreciation of these variables must also include a depot-specific understanding of content and location in order to elucidate mechanisms governing cellular development and regulation of fat deposition. Because adipose tissue depots contain various cell types, differences in the cellularity among and within adipose depots are presently being documented to ascertain functional differences. This has led to the possibility of there being, within any one adipose depot, cellular distinctions that essentially result in adipose depots within depots. The papers comprising this issue will underscore numerous differences in cellularity (development, histogenesis, growth, metabolic function, regulation) of different adipose depots. Such information is useful in deciphering adipose depot involvement both in normal physiology and in pathology. Obesity, diabetes, metabolic syndrome, carcass composition of meat animals, performance of elite athletes, physiology/pathophysiology of aging, and numerous other diseases might be altered with a greater understanding of adipose depots and the cells that comprise them—including stem cells—during initial development and subsequent periods of normal/abnormal growth into senescence. Once thought to be dormant and innocuous, the adipocyte is emerging as a dynamic and influential cell and research will continue to identify complex physiologic regulation of processes involved in adipose depot physiology. PMID:26317047

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a cancer-related human virus, classified as a member of the Gammaherpesvirinae subfamily. We report here the construction of a dual fluorescent-tagged KSHV genome (BAC16-mCherry-ORF45), which constitutively expresses green fluorescent protein (GFP) and contains the tegument multifunctional ORF45 protein as a fusion protein with monomeric Cherry fluorescent protein (mCherry). We confirmed that this virus is properly expressed and correctly replicates and that the mCherry-ORF45 protein is incorporated into the virions. Using this labeled virus, we describe the dynamics of mCherry-ORF45 expression and localization in newly infected cells as well as in latently infected cells undergoing lytic induction and show that mCherry can be used to monitor cells undergoing the lytic viral cycle. This virus is likely to enable future studies monitoring the dynamics of viral trafficking and tegumentation during viral ingress and egress. IMPORTANCE The present study describes the construction and characterization of a new recombinant KSHV genome BAC16 clone which expresses mCherry-tagged ORF45. This virus enables the tracking of cells undergoing lytic infection and can be used to address issues related to the trafficking and maturation pathways of KSHV virions. PMID:25165104

Hydroponic experiments were conducted to investigate the tolerance and spatial distribution of lead (Pb) in Elsholtzia splendens-a copper (Cu) accumulator plant using synchrotron-based micro-X-ray fluorescence. According to chlorophyll concentration and chlorophyll fluorescence parameters, E. splendens displayed certain tolerance at 100 μM Pb treatment. Lead concentration in roots, stems and leaves of E. splendens reached 45,183.6, 1657.6, and 380.9 mg kg(-1), respectively. Pb was mostly accumulated in the roots, and there were also high concentrations of Pb been transported into stems and leaves. Micro-XRF analysis of the stem and leaf cross section revealed that Pb was mostly restricted in the vascular bundles and epidermis tissues of both stem and leaf of E. splendens. The correlation between distribution of K, Ca, Zn and Pb were analyzed. There were significant positive correlations (P<0.01) among Pb and Ca, K, Zn distribution both in stem and leaf of E. splendens. However, among the three elements, Ca shared the most similar distribution pattern and the highest correlation coefficients with Pb in both stem and leaf cross section of E. splendens. This suggests that Ca may play an important role in Pb accumulation in stem and leaf of E. splendens.

Is cholesterol distributed among intracellular compartments by passive equilibration down its chemical gradient? If so, its distribution should reflect the relative cholesterol affinity of the constituent membrane phospholipids as well as their capacity for association with the sterol. We examined this issue by analyzing the reactivity to cholesterol oxidase of large unilamellar vesicles (LUVs) containing phospholipids and varied levels of cholesterol. The rates of cholesterol oxidation differed among the various phospholipid environments by roughly 4 orders of magnitude. Furthermore, accessibility to the enzyme increased by orders of magnitude at cholesterol thresholds that suggested cholesterol:phospholipid association ratios of 1:1, 2:3, or 1:2 (moles:moles). The accessibility of cholesterol above these thresholds was still constrained by its particular phospholipid environment. One phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine, exhibited no threshold. The analysis suggested values for the stoichiometries of the putative cholesterol-phospholipid complexes, their relative stabilities, and the fractions of bilayer cholesterol not in complexes at the threshold equivalence points. Predictably, the saturated phosphorylcholine species had the lowest apparent stoichiometric ratios and the strongest associations with cholesterol. These results are in general agreement with the equilibrium distribution of cholesterol between the various LUVs and methyl-β-cyclodextrin. In addition, the behavior of the cholesterol in intact human red blood cells matched predictions made from LUVs of the corresponding composition. These results support a passive mechanism for the intracellular distribution of cholesterol that can provide a signal for its homeostatic regulation.

Does cholesterol distribute among intracellular compartments by passive equilibration down its chemical gradient? If so, its distribution should reflect the relative cholesterol affinity of the constituent membrane phospholipids as well as their ability to form stoichiometric cholesterol complexes. We tested this hypothesis by analyzing the reactivity to cholesterol oxidase of large unilamellar vesicles (LUVs) containing biological phospholipids plus varied cholesterol. The rates of cholesterol oxidation differed among the various phospholipid environments by roughly four orders of magnitude. Furthermore, accessibility to the enzyme increased by orders of magnitude at cholesterol thresholds that suggested stoichiometries of association of 1:1, 2:3 or 1:2 cholesterol:phospholipid (mol:mol). Cholesterol accessibility above the threshold was still constrained by its particular phospholipid environment. One phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine, exhibited no threshold. The analysis suggested values for the relative stabilities of the cholesterol-phospholipid complexes and for the fractions of bilayer cholesterol not in complexes at the threshold equivalence points; predictably, the saturated phosphorylcholine species had the lowest stoichiometries and the strongest affinities for cholesterol. These results were in general agreement with the equilibrium distribution of cholesterol between the various LUVs and methyl-β-cyclodextrin. In addition, the properties of the cholesterol in intact human red blood cells matched predictions made from LUVs of the corresponding composition. These results support a passive mechanism for the intracellular distribution of cholesterol that can provide a signal for its homeostatic regulation. PMID:24000774

The human immunodeficiency virus type 1 (HIV-1) Tat protein, which is essential for HIV gene expression and viral replication, is known to mediate pleiotropic effects on various cell functions. For instance, Tat protein is able to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction. To study the effect that HIV-1 Tat exerts on the host cell, we identified several genes that were up- or down-regulated in tat-expressing cell lines by using the differential display method. HIV-1 Tat specifically increases the expression of the cleavage and polyadenylation specificity factor (CPSF) 73-kDa subunit (CPSF3) without affecting the expression of the 160- and 100-kDa subunits of the CPSF complex. This complex comprises four subunits and has a key function in the 3'-end processing of pre-mRNAs by a coordinated interaction with other factors. CPSF3 overexpression experiments and knockdown of the endogenous CPSF3 by mRNA interference have shown that this subunit of the complex is an important regulatory protein for both viral and cellular gene expression. In addition to the known CPSF3 function in RNA polyadenylation, we also present evidence that this protein exerts transcriptional activities by repressing the mdm2 gene promoter. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus increases mRNA processing, causing an increase in both cell and viral gene expression.

Expression analysis of transporters selective for monocarboxylates such as lactate and ketone bodies in the kidney contributes to understanding the renal energy metabolism. Distribution and expression intensity of a sodium-dependent monocarboxylate transporter (SMCT) and proton-coupled monocarboxylate transporters (MCT) were examined in the mouse kidney. In situ hybridization survey detected significant mRNA expressions of SMCT and MCT-1, 2, 5, 8, 9, 10, and 12. Among these, signals for SMCT, MCT2 and MCT8 were predominant; transcripts of SMCT were restricted to the cortex and the outer stripe of outer medulla, while those of MCT2 and MCT8 gathered in the inner stripe of outer medulla and the cortex, respectively. Immunohistochemically, SMCT was present at the brush border in S2 and S3 of proximal tubules, suggesting the active uptake of luminal monocarboxylates here. MCT1 and MCT2 immunoreactivities were respectively found baso-laterally in S1 and thick ascending limbs of Henle's loop. The cellular localization of transporters suggests the involvement of SMCT in the uptake of filtrated lactate and ketone bodies and that of MCTs in the transport of monocarboxylate metabolites between tubular cells and circulation, but the different distribution patterns do not support the notion of a functional linkage between SMCT and MCT1/MCT2.

Objective: Dystrophin is part of a protein complex that connects the cytoskeleton to the extracellular matrix. In addition to its role in muscle tissue, it functions as an anchoring protein within the central nervous system such as in hippocampus and cerebellum. Its presence in the latter regions is illustrated by the cognitive problems seen in Duchenne Muscular Dystrophy (DMD). Since epilepsy is also supposed to constitute a comorbidity of DMD, it is hypothesized that dystrophin plays a role in neuronal excitability. Here, we aimed to study brain dystrophin distribution and expression in both, human and experimental temporal lobe epilepsy (TLE). Method: Regional and cellular dystrophin distribution was evaluated in both human and rat hippocampi and in rat cerebellar tissue by immunofluorescent colocalization with neuronal (NeuN and calbindin) and glial (GFAP) markers. In addition, hippocampal dystrophin levels were estimated by Western blot analysis in biopsies from TLE patients, post-mortem controls, amygdala kindled (AK)-, and control rats. Results: Dystrophin was expressed in all hippocampal pyramidal subfields and in the molecular-, Purkinje-, and granular cell layer of the cerebellum. In these regions it colocalized with GFAP, suggesting expression in astrocytes such as Bergmann glia (BG) and velate protoplasmic astrocytes. In rat hippocampus and cerebellum there were neither differences in dystrophin positive cell types, nor in the regional dystrophin distribution between AK and control animals. Quantitatively, hippocampal full-length dystrophin (Dp427) levels were about 60% higher in human TLE patients than in post-mortem controls (p < 0.05), whereas the level of the shorter Dp71 isoform did not differ. In contrast, AK animals showed similar dystrophin levels as controls. Conclusion: Dystrophin is ubiquitously expressed by astrocytes in the human and rat hippocampus and in the rat cerebellum. Hippocampal full-length dystrophin (Dp427) levels are upregulated

We studied the molecular mechanism of successive multistep cervical carcinogenic progression with our previously established in vitro model system. This system was composed of primary human endocervical cells (HEN), two lines of HEN immortalized by HPV16 and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). The expression was examined of diverse cellular genes associated with oncogenesis and senescence, especially for cervical cancer. Consistent results were seen for the pairs of immortalized and malignantly transformed lines. Immortalization of HEN by HPV16 resulted in enhanced expression of H-ras, c-myc, B-myb, p53, p16INK4 and PCNA mRNA; enhanced expression of p16 and PCNA proteins; decreased expression of WAF1/p21/Cip1/Sid1 and fibronectin mRNA; and decreased p53 protein. On the other hand, the CSC-transformed counterparts of HPV16-immortalized cells had up-regulated levels of B-myb, p53 and WAF1 mRNA and p53 protein. Our results indicate that the differential activation or inactivation of multiple cellular genes is important for the immortalization, as well as the transformation, of human cervical cells. Further, we suggest that our in vitro model system is useful for investigating the molecular mechanism of multistep cervical carcinogenesis.

Asthma and chronic obstructive pulmonary disease (COPD) are associated with Th2 and Th1 differentiated T cells. The cytokine thymic stromal lymphopoietin (TSLP) promotes differentiation of Th2 T cells and secretion of chemokines which preferentially attract them. We hypothesized that there is distinct airways expression of TSLP and chemokines which preferentially attract Th1- and Th2-type T cells, and influx of T cells bearing their receptors in asthma and COPD. In situ hybridization, immunohistochemistry, and ELISA were used to examine the expression and cellular provenance of TSLP, Th2-attracting (TARC/CCL17, MDC/CCL22, I-309/CCL1), and Th1-attracting (IP-10/CXCL10, I-TAC/CXCL11) chemokines in the bronchial mucosa and bronchoalveolar lavage fluid of subjects with moderate/severe asthma, COPD, and controls. Cells expressing mRNA encoding TSLP, TARC/CCL17, MDC/CCL22, and IP-10/CXCL10, but not I-TAC/CXCL11 and I-309/CCL1, were significantly increased in severe asthma and COPD as compared with non-smoker controls (p < 0.02). This pattern was reflected in bronchoalveolar lavage fluid protein concentrations. Expression of the same chemokines was also increased in ex- and current smokers. The cellular sources of TSLP and chemokines were strikingly similar in severe asthma and COPD. The numbers of total bronchial mucosal T cells expressing the chemokine receptors CCR4, CCR8, and CXCR3 did not significantly differ in asthma, COPD, and controls. Both asthma and COPD are associated with elevated bronchial mucosal expression of TSLP and the same Th1- and Th2-attracting chemokines. Increased expression of these chemokines is not, however, associated with selective accumulation of T cells bearing their receptors.

To examine the expression of the cellular homolog of the Abelson murine leukemia virus transforming gene (the v-abl sequence), a DNA probe representing the v-abl sequence was prepared. The probe detected two cytoplasmic polyadenylic acid-containing c-abl RNAs of about 6.5 and 5.5 kilobases in a variety of rodent cells, and slightly larger RNAs were detected in human cells. These two RNA species were found in all normal tissues or cell lines examined, but at differing concentrations: liver cells had the least, fibroblastic cell lines had the most. By using a probe able to detect the cellular but not the viral gene, the two RNAs were shown to be present in Abelson murine leukemia virus-transformed cells at levels found either in their untransformed counterparts or in similar cell types transformed by other means. The target cells of the virus have a somewhat elevated level of the two RNAs although expression of the c-abl gene is not restricted to these cells. The v-abl sequence lacks 0.35 and 0.85 kilobases of the c-abl RNA on the 5' and 3' ends, respectively. Thus, the Abelson murine leukemia virus transforming gene is an internal fragment of the transcript of a normal cellular gene. Images PMID:6306446

Cellular senescence is an irreversible cell cycle arrest that limits the replicative lifespan of cells. Senescence suppresses development of tumors by regulating aging factors, such as cyclin dependent kinase inhibitor (CKI) and telomerase. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed between young human mesenchymal stem cells (Y-hMSCs) and senescent human mesenchymal stem cells (S-hMSCs). We selected positive clones that were functionally characterized by referring to public databases using NCBI BLAST tool. This search revealed that 19 genes were downregulated, and 43 genes were upregulated in S-hMSCs relative to Y-hMSCs. Among subtracted clones in Y-hMSCs, most of genes markedly were related to metabolic functions. These genes, PDIA3, WDR1, FSTL1, COPG1, LMAN1, and PDIA6, significantly downregulated. Conversely, genes for subtracted clones in S-hMSCs were mostly associated with cell adhesion. In particular, the expression levels of 9 genes, HSP90B1, EID1, ATP2B4, DDAH1, PRNP, RAB1A, PGS5, TM4SF1 and SSR3, gradually increased during senescence. These genes have not previously been identified as being related to cellular senescence, but they seemed to be potentially affected during cellular senescence.

We report a facile approach for the fabrication of a new class of graphene oxide (GO)-based nanoassemblies by layer-by-layer (LbL) technique. The single-layer thickness and intrinsic negatively charged carboxyl groups of GO nanosheets provide a natural platform for LbL assembly of polyelectrolyte nanofilms by electrostatic forces at mild and aqueous conditions. The general applicability of our approach is demonstrated by the preparation of GO nanoassemblies with sizes of 100-200 nm using various charged polyelectrolytes, including synthetic polymers, polypeptides, and DNA oligonucleotides. Systemic assessment of cytotoxicity and acute stress response show that no discernable signs of cytotoxicity are associated with exposure of GO and its nanoassemblies [GO/PLL (poly ( l-lysine)), GO/PLL/PSS (poly(sodium-4-styrenesulfonate)), GO/PLL-PEG (PEGlayted PLL), GO/PLL/PLGA-PEG (PEGlayted poly ( l-glutamic acid))] up to 1 μg/mL. Studies on cellular uptake and subcellular localization show that a representative nanoassembly, GO/PLL-PEG, can effectively cross cell membranes and localize mainly in lysosomal compartments, without induction of noticeable harmful effects as confirmed by detection of mitochondrial depolarization and lysosomal pH.

Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.

Both positive feedback learning and negative feedback learning are essential for adapting and optimizing behavioral performance. There is increasing evidence in humans and animals that the ability of negative feedback learning emerges postnatally. Our work in rats, using a two-way active avoidance task (TWA) as an experimental paradigm for negative feedback learning, revealed that medial and lateral prefrontal regions of infant rats undergo dramatic synaptic reorganization during avoidance training, resulting in improved avoidance learning in adulthood. The aim of this study was to identify changes of cellular activation patterns during the course of training and in relation to infant pretraining. We applied a quantitative cellular imaging technique using the immunocytochemical detection of the activity marker early growth response protein 1 (Egr1) as a candidate contributing to learning-induced synaptic plasticity. We found region-specific cellular activity patterns, which indicate that during the acquisition phase, Egr1 expression is specifically elevated in cellular ensembles of the orbitofrontal, dorsal anterior cingulate and hippocampal CA1 region. During memory retrieval Egr1 expression is elevated in cellular ensembles of the dentate gyrus. Moreover, we, for the first time, show here that TWA training during infancy alters adult learning- and memory-related patterns of Egr1 expression in these brain regions. It is tempting to speculate that during infant learning, specific Egr1-expressingcellular ensembles are "tagged" representing long-term memory formation, and that these cell ensembles may be reactivated during adult learning.

Defects of adrenoleukodystrophy protein (ALDP) lead to X-linked adrenoleukodystrophy (X-ALD), a disorder mainly affecting the nervous system white matter and the adrenal cortex. In the present study, we examine the expression of ALDP in various human tissues and cell lines by multiple-tissue RNA expression array analysis, Western blot analysis, and immunohistochemistry. ALDP-encoding mRNA is most abundant in tissues with high energy requirements such as heart, muscle, liver, and the renal and endocrine systems. ALDP selectively occurs in specific cell types of brain (hypothalamus and basal nucleus of Meynert), kidney (distal tubules), skin (eccrine gland, hair follicles, and fibroblasts), colon (ganglion cells and epithelium), adrenal gland (zona reticularis and fasciculata), and testis (Sertoli and Leydig cells). In pituitary gland, ALDP is confined to adrenocorticotropin-producing cells and is significantly reduced in individuals receiving long term cortisol treatment. This might indicate a functional link between ALDP and proopiomelanocortin-derived peptide hormones.

Quantitative structure-activity relationship (QSAR) studies and mechanistic mathematical modeling approaches have been independently employed for analyzing and predicting the transport and distribution of small molecule chemical agents in living organisms. Both of these computational approaches have been useful to interpret experiments measuring the transport properties of small molecule chemical agents, in vitro and in vivo. Nevertheless, mechanistic cell-based pharmacokinetic models have been especially useful to guide the design of experiments probing the molecular pathways underlying small molecule transport phenomena. Unlike QSAR models, mechanistic models can be integrated from microscopic to macroscopic levels, to analyze the spatiotemporal dynamics of small molecule chemical agents from intracellular organelles to whole organs, well beyond the experiments and training data sets upon which the models are based. Based on differential equations, mechanistic models can also be integrated with other differential equations-based systems biology models of biochemical networks or signaling pathways. Although the origin and evolution of mathematical modeling approaches aimed at predicting drug transport and distribution has occurred independently from systems biology, we propose that the incorporation of mechanistic cell-based computational models of drug transport and distribution into a systems biology modeling framework is a logical next-step for the advancement of systems pharmacology research. PMID:24218242

During cellular reprogramming only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of Fbxo15, Fgf4, and Oct4 previously suggested to be reprogramming markers. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc and Nanog, can activate the pluripotency circuitry. PMID:22980981

Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme-mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer’s disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron-loaded oligodendrocytes in the basal ganglia and thalamus of Hx-null mice compared to wild-type controls. Interestingly, there was no increase in H- and L-ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer’s disease and Parkinson’s disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx-null animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu–Zn superoxide dismutase-1 expression were higher in the brain of Hx-null mice than in that of wild-type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases. PMID:19120692

Abstract Heart diseases are major causes of morbidity and mortality in Western society. Gene therapy approaches are becoming promising therapeutic modalities to improve underlying molecular processes affecting failing cardiomyocytes. Numerous cardiac clinical gene therapy trials have yet to demonstrate strong positive results and advantages over current pharmacotherapy. The success of gene therapy depends largely on the creation of a reliable and efficient delivery method. The establishment of such a system is determined by its ability to overcome the existing biological barriers, including cellular uptake and intracellular trafficking as well as modulation of cellular permeability. In this article, we describe a variety of physical and mechanical methods, based on the transient disruption of the cell membrane, which are applied in nonviral gene transfer. In addition, we focus on the use of different physiological techniques and devices and pharmacological agents to enhance endothelial permeability. Development of these methods will undoubtedly help solve major problems facing gene therapy. PMID:23427834

Innexins are a family of membrane proteins involved in the formation of gap junctions in invertebrates. They have been found to participate in several aspects of cell differentiation and in embryonic patterning through the formation of specific intercellular communication channels. We present here data showing that the recently identified innexin of the marine worm Chaetopterus variopedatus is expressed only in particular cells of the early stage, demonstrating cell specificity of innexin expression also in polychaete annelids. Phylogenetic analysis of all known innexins results in a phylogenetic tree clearly distinguishing insect, nematode, and other invertebrate innexins. Comparative analysis of proteins and known related genes shows that the apparent similarity of protein composition, overall structural organization, and specificity of cellularexpression, typical of innexins of all studied organisms, correspond to highly heterogeneous gene structures even for genes that are in close contiguity on the same chromosome. A possible evolutionary motive producing this situation is discussed.

Recombinant measles viruses (rMV) based on the live attenuated measles vaccine strain (MVb) expressing antigens of HIV-1 clade B were generated by reverse genetics. Recombinants expressing single or double antigens of HIV-1 (rMV-HIV) were genetically highly stable on human diploid cells. The production process of these viruses was essentially similar to the parental MV strain, yielding comparative end titers. Immunization of tg-mice by different regimens and formulations showed potent humoral and cellular immune responses against MV and HIV antigens. Recombinant MV-HIV expressing Gag protein conferred protective immunity in tg-mice after a high-dose pseudochallenge with recombinant vaccinia virus. In addition, rMV-HIV boosted anti-HIV antibodies, in the presence of pre-existing anti-vector antibodies.

Neurotrophins and their receptors modulate cerebral cortical development. Tubers in the tuberous sclerosis complex (TSC) are characterized histologically by disorganized cortical cytoarchitecture and thus, we hypothesized that expression of neurotrophin mRNAs and proteins might be altered in tubers. Using in situ transcription and mRNA amplification to probe cDNA arrays, we found that neurotrophin-3 (NT3) and trkB mRNA expression were reduced whereas neurotrophin-4 (NT4) and trkC mRNA expression were increased in whole tuber sections. Alterations in mRNA abundance were defined in single microdissected dysplastic neurons (DNs) and giant cells (GCs). NT3 mRNA expression was reduced in GCs and trkB mRNA expression was reduced in DNs. NT4 mRNA expression was increased in DNs and trkC mRNA expression was increased in both DNs and GCs. In three patients, TSC2 locus mutations were confirmed and the mean tuberin mRNA expression levels was reduced across all nine cases. Consistent with these observations, NT3 mRNA expression was reduced but trkC mRNA expression was increased in vitro in human NTera2 neurons (NT2N) transfected with a tuberin antisense construct that reduced tuberin expression. Western analysis of tuber homogenates and computer-assisted densitometry of immunolabeled sections confirmed the neurotrophin mRNA expression data in whole sections and single neurotrophin immunoreactive cells. We conclude that alterations in NT4/trkB and NT3/trkC expression may contribute to tuber formation during brain development as downstream effects of the hamartin and tuberin pathway in TSC.

Methods developed based on bifurcation theory have demonstrated their potential in driving network identification for complex human diseases, including the work by Chen, et al. Recently bifurcation theory has been successfully applied to model cellular differentiation. However, there one often faces a technical challenge in driving network prediction: time course cellular differentiation study often only contains one sample at each time point, while driving network prediction typically require multiple samples at each time point to infer the variation and interaction structures of candidate genes for the driving network. In this study, we investigate several methods to identify both the critical time point and the driving network through examination of how each time point affects the autocorrelation and phase locking. We apply these methods to a high-throughput sequencing (RNA-Seq) dataset of 42 subsets of thymocytes and mature peripheral T cells at multiple time points during their differentiation (GSE48138 from GEO). We compare the predicted driving genes with known transcription regulators of cellular differentiation. We will discuss the advantages and limitations of our proposed methods, as well as potential further improvements of our methods.

Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr2,6,11,15]-Gm, and [Ser2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr2,6,11,15, Pro9]-D-Gm, and [Thr2,6,11,15, D-Pro9]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity. PMID:24312251

Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr(2,6,11,15)]-Gm, and [Ser(2,6,11,15)]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr(2,6,11,15)]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr(2,6,11,15), Pro(9)]-D-Gm, and [Thr(2,6,11,15), D-Pro(9)]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.

Members of the vascular endothelial growth factor (VEGF) family are key signaling proteins in the induction and regulation of angiogenesis, both during development and in pathological conditions. However, signaling mediated through VEGF family proteins and their receptors has recently been shown to have direct effects on neurons and glial cells. In the present study, we immunocytochemically investigated the expression and cellulardistribution of VEGFA, VEGFB, and their associated receptors (VEGFR-1 and VEGFR-2) in focal cortical dysplasia (FCD) type IIB from patients with medically intractable epilepsy. Histologically normal temporal cortex and perilesional regions displayed neuronal immunoreactivity (IR) for VEGFA, VEGFB, and VEGF receptors (VEGFR-1 and VEGFR-2), mainly in pyramidal neurons. Weak IR was observed in blood vessels and there was no notable glial IR within the grey and white matter. In all FCD specimens, VEGFA, VEGFB, and both VEGF receptors were highly expressed in dysplastic neurons. IR in astroglial and balloon cells was observed for VEGFA and its receptors. VEGFR-1 displayed strong endothelial staining in FCD. Double-labeling also showed expression of VEGFA, VEGFB and VEGFR-1 in cells of the microglia/macrophage lineage. The neuronal expression of both VEGFA and VEGFB, together with their specific receptors in FCD, suggests autocrine/paracrine effects on dysplastic neurons. These autocrine/paracrine effects could play a role in the development of FCD, preventing the death of abnormal neuronal cells. In addition, the expression of VEGFA and its receptors in glial cells within the dysplastic cortex indicates that VEGF-mediated signaling could contribute to astroglial activation and associated inflammatory reactions. PMID:18317782

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. 125I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with Mr 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (Mr 39,000, 88,000), stems (Mr 45,000, 60,000, 64,000), and roots (Mr 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. 125I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with Mr 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (Mr 39,000, 88,000), stems (Mr 45,000, 60,000, 64,000), and roots (Mr 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.

The therapeutic application of ionizing radiation is associated with thrombotic events, but the exact underlying molecular mechanisms are still unclear. Tissue factor (TF), the primary initiator of blood coagulation, is essentially involved in the pathophysiology of thrombosis. Circulating monocytes have been identified to upregulate TF under inflammatory conditions and, thereby, enhance blood thrombogenicity. The study examines the effect of irradiation on the cellular procoagulability and TF protein expression of human peripheral blood mononuclear cells (PBMNCs) in a time period of 7 days. Human PBMNCs were irradiated with 20 Gy. Procoagulability of PBMNCs, released microparticles and microparticle-free cell supernatant was analyzed by a chromogenic assay and TF protein expression quantified by TF ELISA. To determine whether irradiated PBMNCs and shed microparticles initiate plasma clotting, a one stage clotting assay was performed. We found a significant increase of PBMNC-associated procoagulant activity over a time period of 7 days post irradiation. Moreover, 3 days post irradiation PBMNCs initiated the plasma clotting faster than non-irradiated cells. An enhanced cellular TF protein concentration was persistently observed throughout the investigated time up to 7 days post irradiation. Microparticle-associated TF activity significantly increased 3 days post irradiation compared with the non-irradiated controls. PBMNC-derived microparticles post irradiation also initiated the plasma clotting faster than microparticles derived from controls. The results show irradiation to induce TF expression and to increase procoagulability of PBMNCs and cell-derived microparticles. This could be a possible mechanism by which ionizing radiation enhances blood thrombogenicity.

Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments. PMID:27729845

Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments.

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric β-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat. PMID:10069821

Tumour hypoxia promotes radioresistance and is associated with poor prognosis. The transcription factor Aryl hydrocarbon receptor nuclear translocator (ARNT), also designated as Hypoxia-inducible factor (HIF)-1β, is part of the HIF pathway which mediates cellular adaptations to oxygen deprivation and facilitates tumour progression. The subunits HIF-1α and ARNT are key players within this pathway. HIF-1α is regulated in an oxygen-dependent manner whereas ARNT is considered to be constitutively expressed. However, there is mounting evidence that certain tumour cells are capable to elevate ARNT in hypoxia which suggests a survival benefit. Therefore the objective of this study was to elucidate effects of an altered ARNT expression level on the cellular response to radiation. Different human cell lines (Hep3B, MCF-7, 786-Owt, 786-Ovhl, RCC4wt and RCC4vhl) originating from various tumour entities (Hepatocellular carcinoma, breast cancer and renal cell carcinoma respectively) were X-irradiated using a conventional linear accelerator. Knockdown of ARNT expression was achieved by transient siRNA transfection. Complementary experiments were performed by forced ARNT overexpression using appropriate plasmids. Presence/absence of ARNT protein was confirmed by Western blot analysis. Clonogenic survival assays were performed in order to determine cellular survival post irradiation. Statistical comparison of two groups was achieved by the unpaired t-test. The results of this study indicate that ARNT depletion renders tumour cells susceptible to radiation whereas overexpression of this transcription factor confers radioresistance. These findings provide evidence to consider ARNT as a drug target and as a predictive marker in clinical applications concerning the response to radiation.

Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarray technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML.

While cellular LC3B and SQSTM1 levels serve as key autophagy markers, their regulation by different signaling pathways requires better understanding. Here, we report the mechanisms by which the Raf/MEK/ERK pathway regulates cellular LC3B and SQSTM1 levels. In different cell types, δRaf-1:ER- or B-RafV600E-mediated MEK/ERK activation increased LC3B-I, LC3B-II, and SQSTM1/p62 levels, which was accompanied by increased BiP/GRP78 expression. Use of the autophagy inhibitors chloroquine and bafilomycin A1, or RNA interference of ATG7, suggested that these increases in LC3B and SQSTM1 levels were in part attributed to altered autophagic flux. However, intriguingly, these increases were also attributed to their increased expression. Upon Raf/MEK/ERK activation, mRNA levels of LC3B and SQSTM1 were also increased, and subsequent luciferase reporter analyses suggested that SQSTM1 upregulation was mediated at transcription level. Under this condition, transcription of BiP/GRP78 was also increased, which was necessary for Raf/MEK/ERK to regulate LC3B at the protein, but not mRNA, level. This suggests that BiP has a role in regulating autophagy machinery when Raf/MEK/ERK is activated. In conclusion, these results suggest that, under a Raf/MEK/ERK-activated condition, the steady-state cellular levels of LC3B and SQSTM1 can also be determined by their altered expression wherein BiP is utilized as an effector of the signaling. PMID:25128814

The distribution in the rat nervous system of acidic and basic fibroblast growth factors (FGFs) was analysed by a combination of biochemical and anatomical methods. Acidic FGF (aFGF) was found to be present exclusively in specific neuronal populations, such as motor neurons and basal forebrain cholinergic neurons. Basic FGF (bFGF) was found in astrocytes and in neurons in hippocampal area CA2. Within labelled astrocytes and CA2-neurons, bFGF was detected in both the cytoplasm and the nucleus. The levels of intracellular bFGF were manipulated by antisense oligonucleotide treatment of cultures of developing neural crest cells. Results indicated that the amount of melanogenesis in the cultures is likely to be regulated by intracellular, possibly nuclear bFGF.

Purpose We evaluated the role of a poly(ethylene glycol) (PEG) surface coating to increase residence times and alter the cellular fate of nano- and microparticles delivered to the lung. Methods Three sizes of PRINT hydrogel particles (80×320 nm, 1.5 and 6 um donuts) with and without a surface PEG coating were instilled in the airways of C57/b6 mice. At time points of 1, 7, and 28 days, BALF and whole lungs were evaluated for the inflammatory cytokine Il-6 and chemokine MIP-2, histopathology, cellular populations of macrophages, dendritic cells (DCs), and granulocytes, and particulate uptake within these cells through flow cytometry, ELISAs, and fluorescent imaging. Results Particles of all sizes and surface chemistries were readily observed in the lung with minimal inflammatory response at all time points. Surface modification with PEGylation was found to significantly increase lung residence times and homogeneous lung distribution, delaying macrophage clearance of all sizes, with the largest increase in residence time observed for 80×320 nm particles. Additionally, it was observed that DCs were recruited to the airway following administration of unPEGylated particles and preferentially associated with these particles. Conclusions Pulmonary drug delivery vehicles designed with a PEG surface coating can be used to delay particle uptake and promote cell-specific targeting of therapeutics. PMID:26002743

RNA silencing, mediated by small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs), is a potent antiviral or antibacterial mechanism, besides regulating normal cellular gene expression critical for development and physiology. To gain insights into host small RNA metabolism under infections by different viruses, we used Solexa/Illumina deep sequencing to characterize the small RNA profiles of rice plants infected by two distinct viruses, Rice dwarf virus (RDV, dsRNA virus) and Rice stripe virus (RSV, a negative sense and ambisense RNA virus), respectively, as compared with those from non-infected plants. Our analyses showed that RSV infection enhanced the accumulation of some rice miRNA*s, but not their corresponding miRNAs, as well as accumulation of phased siRNAs from a particular precursor. Furthermore, RSV infection also induced the expression of novel miRNAs in a phased pattern from several conserved miRNA precursors. In comparison, no such changes in host small RNA expression was observed in RDV-infected rice plants. Significantly RSV infection elevated the expression levels of selective OsDCLs and OsAGOs, whereas RDV infection only affected the expression of certain OsRDRs. Our results provide a comparative analysis, via deep sequencing, of changes in the small RNA profiles and in the genes of RNA silencing machinery induced by different viruses in a natural and economically important crop host plant. They uncover new mechanisms and complexity of virus-host interactions that may have important implications for further studies on the evolution of cellular small RNA biogenesis that impact pathogen infection, pathogenesis, as well as organismal development. PMID:21901091

Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17β-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an ‘activator’ or a ‘repressor’ possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. PMID:26295471

We have recently reported that expression in yeast cells of P-glycoprotein (P-gp) encoded by the mouse multidrug resistance mdr3 gene (Mdr3) can complement a null ste6 mutation (M. Raymond, P. Gros, M. Whiteway, and D. Y. Thomas, Science 256:232-234, 1992). Here we show that Mdr3 behaves as a fully functional drug transporter in this heterologous expression system. Photolabelling experiments indicate that Mdr3 synthesized in yeast cells binds the drug analog [125I]iodoaryl azidoprazosin, this binding being competed for by vinblastine and tetraphenylphosphonium bromide, two known multidrug resistance drugs. Spheroplasts expressing wild-type Mdr3 (Ser-939) exhibit an ATP-dependent and verapamil-sensitive decreased accumulation of [3H]vinblastine as compared with spheroplasts expressing a mutant form of Mdr3 with impaired transport activity (Phe-939). Expression of Mdr3 in yeast cells can confer resistance to growth inhibition by the antifungal and immunosuppressive agent FK520, suggesting that this compound is a substrate for P-gp in yeast cells. Replacement of Ser-939 in Mdr3 by a series of amino acid substitutions is shown to modulate both the level of cellular resistance to FK520 and the mating efficiency of yeast mdr3 transformants. The effects of these mutations on the function of Mdr3 in yeast cells are similar to those observed in mammalian cells with respect to drug resistance and transport, indicating that transport of a-factor and FK520 in yeast cells is mechanistically similar to drug transport in mammalian cells. The ability of P-gp to confer cellular resistance to FK520 in yeast cells establishes a dominant phenotype that can be assayed for the positive selection of intragenic revertants of P-gp inactive mutants, an important tool for the structure-function analysis of mammalian P-gp in yeast cells. Images PMID:7505392

Oncogene-induced senescence (OIS) is a critical tumor-suppressing mechanism that restrains cancer progression at premalignant stages, in part by causing telomere dysfunction. Currently it is unknown whether this proliferative arrest presents a stable and therefore irreversible barrier to cancer progression. Here we demonstrate that cells frequently escape OIS induced by oncogenic H-Ras and B-Raf, after a prolonged period in the senescence arrested state. Cells that had escaped senescence displayed high oncogene expression levels, retained functional DNA damage responses, and acquired chromatin changes that promoted c-Myc–dependent expression of the human telomerase reverse transcriptase gene (hTERT). Telomerase was able to resolve existing telomeric DNA damage response foci and suppressed formation of new ones that were generated as a consequence of DNA replication stress and oncogenic signals. Inhibition of MAP kinase signaling, suppressing c-Myc expression, or inhibiting telomerase activity, caused telomere dysfunction and proliferative defects in cells that had escaped senescence, whereas ectopic expression of hTERT facilitated OIS escape. In human early neoplastic skin and breast tissue, hTERT expression was detected in cells that displayed features of senescence, suggesting that reactivation of telomerase expression in senescent cells is an early event during cancer progression in humans. Together, our data demonstrate that cells arrested in OIS retain the potential to escape senescence by mechanisms that involve derepression of hTERT expression. PMID:27503890

The stable expression of the Epstein-Barr virus (EBV) latent membrane protein (LMP) in certain EBV-negative Burkitt's lymphoma cell lines correlates with an increased expression of the oncogene Bcl-2 (S. Henderson, M. Rowe, C. Gregory, D. Croom-Carter, F. Wang, R. Longnecker, E. Kieff, and A. Rickinson, Cell 65:1107-1115, 1991). This finding is consistent with a model in which Bcl-2 contributes to the immortalization of B cells mediated by EBV. We therefore asked whether the expression of Bcl-2 protein correlates with the induction of three cellular phenotypes induced by or associated with LMP. The expression of Bcl-2 in primary B cells infected with the B95-8 strain of EBV varied between 1 and 1.8 times that in uninfected cells when 50% of the cells were infected, expressed LMP, and incorporated 20-fold more [3H]thymidine than did uninfected cells. This finding indicates that induced proliferation of these primary cells is not sufficient to induce Bcl-2. We found that BALB/c 3T3 cells and their derivatives transformed by LMP do not express Bcl-2 detectably. The expression of LMP at high levels in lymphoid cells is cytotoxic and correlates with an increased expression of Bcl-2 following stable selection for the introduced LMP gene; 2 days after transfection, control vector- and LMP-transfected populations, however, express equal levels of Bcl-2 protein. We also analyzed transient expression of LMP in an EBV-negative Burkitt's lymphoma cell line. Infection of BJAB cells with the B95-8 strain of EBV results in an increase in Bcl-2 expression with a time course similar to that of LMP expression, and LMP alone transiently induces an increase in Bcl-2 expression in these cells. We interpret these observations to indicate that increased expression of Bcl-2 is unlikely to contribute to the ability of EBV to immortalize primary B cells and that both the transformation of rodent cells and the cytotoxicity mediated by LMP are independent of Bcl-2. Images PMID:8394449

Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2 degrees C and +6 degrees C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (+/-1 degrees C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2 degrees C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6 degrees C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms.

Background Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology. However, recent studies have identified early cellular dysfunctions that preclude aggregate formation. Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches. Previously, we demonstrated that tricyclic pyrone (TP) compounds efficiently inhibited formation of amyloid-β (Aβ) aggregates in cell and mouse models representing Alzheimer's Disease (AD). In the present study, we aimed to determine whether TP compounds could prevent aggregation and restore early cellular defects in primary embryonic striatal neurons from animal model representing HD. Results TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells. Treatment with TP compounds also alleviated cholesterol accumulation and restored clathrin-independent endocytosis in HD neurons. Conclusion We have found that TP compounds not only blocked mhtt-induced aggregation, but also alleviated early cellular dysfunctions that preclude aggregate formation. Our data suggest TP molecules may be used as lead compounds for prevention or treatment of multiple neurodegenerative diseases including HD and AD. PMID:19586540

Mitogen-activated normal human peripheral blood lymphocytes bind transferrin to specific membrane receptors. In this study, lymphocytes stimulated with phytohaemagglutinin for 0-66 hr were examined to determine the relation of this phenomenon to cellular activation and related metabolic events. Transferrin receptors were first detected at 20-24 hr. This event was consistently preceded by RNA and protein turnover which commenced during the first 6 hr of culture, whereas initiation of DNA synthesis was detected concurrently with the appearance of receptors or slightly later (24-30 hr). Exposure of cells to inhibitors of RNA and protein synthesis early during culture (at 0 or 24 hr) prevented the expression of transferrin receptors, but also caused generalized metabolic failure, and abrogated cellular activation. In contrast, later addition of these agents at 48 hr did not interfere significantly with the process of activation, but did suppress the terminal increase in receptor-bearing cells observed during the final 18 hr in control cultures lacking inhibitor. After deliberate thermal stripping of receptors from activated cells, the reappearance of membrance binding sites which normally occurred within 30 min, was also blocked by cycloheximide, puromycin and actinomycin D. However, similar inhibition of DNA which was induced by hydroxyurea had much less effect upon both the initial appearance of receptors and their reappearance after ligand-induced depletion. These results demonstrate that the appearance of transferrin receptors upon human lymphocytes is dependent upon cellular activation and requires synthesis of protein and RNA. PMID:6172372

Light that is scattered from lenses and mirrors in an optical system produces a halo of stray light around bright objects within the field of view. The angular distribution of scattered light from any one component is usually described by the Harvey model. This paper presents analytic expressions for the scattered irradiance at a focal plane from optical components that scatter light in accordance with the Harvey model. It is found that the irradiance is independent of the location of an optical element within the system, provided the element is not located at or near an intermediate image plane. It is also found that the irradiance has little or no dependence on the size of the element.

Foraminifera, marine unicellular organism, have been considered as one of the major carbonate producer in ocean. Their calcareous tests are broadly utilized as paleo-environmental indicators in various studies of earth science because their tests have been archived as numerous fossil in sediment for long time and various environmental information are brought by population, morphology and geochemical fingerprints. The knowledge about the cytological process on carbonate precipitation has been described for couples of decade using by OM, SEM and TEM. Foraminiferal management of calcium and carbonate ion uptake into foraminiferal tests from ambient seawater are of great interest. Our previous studies showed the potential to understanding the biomineralization of foraminifera by the application of fluorescent indicators. Recently, we visualize the spatial distributions of cytological calcium and pH in living cell at same time. Observed results show that foraminifera controls very detailed timing of pH variation and concentration of calcium at any stage of chamber formation dynamically. These observations results will help to consider how the geochemical compositions arranging on the foraminiferal test, sensitivity of pH proxy of boron and others.

Histochemical studies were performed on tissues from piglets of different ages treated orally with iron dextran soon after birth. The mucosal cells in the distal region of the small intestine were heavily laden with stainable iron granules during the first three days after the iron administration. The absorptive epithelial cells are desquamated within seven to ten days after birth. Consequently, the number of iron granules gradually diminishes during the first seven days after treatment and no iron granules are demonstrated 12 days after the administration of iron. The iron dextran complex is pinocytosed in newborn piglets and then transported via the lymphatic system. Thus the sinusoidal lining cells of the body and mesenteric lymph nodes are already heavily laden with iron granules 24 hours after oral treatment. This iron store is released only slowing during the first weeks of life. Great amounts of iron granules are demonstrated in the liver and spleen macrophages during the first week after the administration of iron. Due to the rapid utilization of iron in growing piglets these iron stores diminish sharply during the weeks following birth. The distribution of stainable iron in the lymph nodes, liver and spleen seven days after intramuscular injection of iron dextran in newborn piglets was comparable to that for oral administration at that stage of the experiment. Images Fig. 1a-e. Fig. 2a-e. Fig. 3a-d. Fig. 4a-d. Fig. 5a-d. PMID:907907

The visual function of the vertebrate retina relies on sufficient supply with oxygen. Neuroglobin is a respiratory protein thought to play an essential role in oxygen homeostasis of neuronal cells. For further understanding of its function, we compared the distribution of neuroglobin and mitochondria in both vascular and avascular mammalian retinae. In the vascular retinae of mouse and rat, oxygen is supplied by the outer choroidal, deep retinal, and inner capillaries. We show that in this type of retina, mitochondria are concentrated in the inner segments of photoreceptor cells, the outer and the inner plexiform layers, and the ganglion cell layer. These are the same regions in which oxygen consumption takes place and in which neuroglobin is present at high levels. In the avascular retina of guinea pig the deep retinal and inner capillaries are absent. Therefore, only the inner segments of the photoreceptors adjacent to choroidal capillaries display an oxidative metabolism. We demonstrate that in the retina of guinea pigs both neuroglobin and mitochondria are restricted to this layer. Our results clearly demonstrate an association of neuroglobin and mitochondria, thus supporting the hypothesis that neuroglobin is a respiratory protein that supplies oxygen to the respiratory chain.

Fibroblast growth factor 19 (FGF19) and FGF21 are members of a subfamily of the FGFs called endocrine FGFs. FGF19 regulates the bile acid synthetic pathway. FGF19 expression is induced by farnesoid X receptor (FXR), a nuclear hormone receptor activated by bile acids in the small intestine. FGF21 plays an important role in lipolysis that occurs in white adipose tissue. FGF21 expression is stimulated by the nuclear fatty acid receptor peroxisome proliferator-activated receptor α (PPARα) in the liver. FGF19 and FGF21 were recently identified as targets of activating transcription factor 4 (ATF4), which is activated in response to endoplasmic reticulum (ER) stress. ATF4 is also activated by oxidative stress and amino acid deprivation. In this study, we investigated FGF19 and FGF21 expression in response to oxidative stress and amino acid deprivation. We found that FGF19 mRNA is induced by oxidative stress inducers in Caco-2 cells, which are derived from the human intestinal epithelium, and rat intestinal epithelial IEC6 cells. In contrast, ileal FGF15 expression, the rodent ortholog of human FGF19, is not increased by oxidative stress. No notable changes in expression of FGF15/19 took place under amino acid deprivation either in vitro or in vivo. In contrast, FGF21 expression is induced by oxidative stress and amino acid deprivation both in vitro and in vivo. These results indicate distinctive patterns of regulation of FGF19 expression by ER stress, and FGF21 expression by ER stress, oxidative stress, and amino acid deprivation through ATF4 activation.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene predispose to the development of pituitary adenomas. Here, we characterized AIP mutation positive (AIPmut+) and AIP mutation negative (AIPmut-) pituitary adenomas by immunohistochemistry. The expressions of the AIP-related proteins aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), cyclin-dependent kinase inhibitor 1B encoding p27(Kip1), and hypoxia-inducible factor 1-alpha were examined in 14 AIPmut+ and 53 AIPmut- pituitary adenomas to detect possible expression differences. In addition, the expression of CD34, an endothelial and hematopoietic stem cell marker, was analyzed. We found ARNT to be less frequently expressed in AIPmut+ pituitary adenomas (P = 0.001), suggesting that AIP regulates the ARNT levels. AIP small interfering RNA-treated HeLa, HEK293, or Aip-null mouse embryonic fibroblast cells did not show lowered expression of ARNT. Instead, in the pituitary adenoma cell line GH3, Aip silencing caused a partial reduction of Arnt and a clear increase in cell proliferation. We also observed a trend for increased expression of nuclear AHR in AIPmut+ samples, although the difference was not statistically significant (P = 0.06). The expressions of p27(Kip1), hypoxia-inducible factor 1-alpha, or CD34 did not differ between tumor types. The present study shows that the expression of ARNT protein is significantly reduced in AIPmut+ tumors. We suggest that the down-regulation of ARNT may be connected to an imbalance in AHR/ARNT complex formation arising from aberrant cAMP signaling.

Homeobox genes are a family of transcription factors that play a pivotal role in embryogenesis. Prospero homeobox 1 (PROX1) has been shown to function as a tumor suppressor gene or oncogene in various types of cancer, including oral squamous cell carcinoma (OSCC). We have previously identified PROX1 as a downregulated gene in OSCC. The aim of this study is to clarify the underlying mechanism by which PROX1 regulates tumorigenicity of OSCC cells. PROX1 mRNA and protein expression levels were first investigated in 40 samples of OSCC and in nontumor margins. Methylation and amplification analysis was also performed to assess the epigenetic and genetic mechanisms involved in controlling PROX1 expression. OSCC cell line SCC9 was also transfected to stably express the PROX1 gene. Next, SCC9-PROX1-overexpressing cells and controls were subjected to proliferation, differentiation, apoptosis, migration, and invasion assays in vitro. OSCC samples showed reduced PROX1 expression levels compared with nontumor margins. PROX1 amplification was associated with better overall survival. PROX1 overexpression reduces cell proliferation and downregulates cyclin D1. PROX1-overexpressing cells also exhibited reduced CK18 and CK19 expression and transcriptionally altered the expression of WISP3, GATA3, NOTCH1, and E2F1. Our results suggest that PROX1 functions as a tumor suppressor gene in oral carcinogenesis.

Abstract Homeobox genes are a family of transcription factors that play a pivotal role in embryogenesis. Prospero homeobox 1 (PROX1) has been shown to function as a tumor suppressor gene or oncogene in various types of cancer, including oral squamous cell carcinoma (OSCC). We have previously identified PROX1 as a downregulated gene in OSCC. The aim of this study is to clarify the underlying mechanism by which PROX1 regulates tumorigenicity of OSCC cells. PROX1 mRNA and protein expression levels were first investigated in 40 samples of OSCC and in nontumor margins. Methylation and amplification analysis was also performed to assess the epigenetic and genetic mechanisms involved in controlling PROX1 expression. OSCC cell line SCC9 was also transfected to stably express the PROX1 gene. Next, SCC9-PROX1-overexpressing cells and controls were subjected to proliferation, differentiation, apoptosis, migration, and invasion assays in vitro. OSCC samples showed reduced PROX1 expression levels compared with nontumor margins. PROX1 amplification was associated with better overall survival. PROX1 overexpression reduces cell proliferation and downregulates cyclin D1. PROX1-overexpressing cells also exhibited reduced CK18 and CK19 expression and transcriptionally altered the expression of WISP3, GATA3, NOTCH1, and E2F1. Our results suggest that PROX1 functions as a tumor suppressor gene in oral carcinogenesis. PMID:25526434

The role of astrocytes in HIV-1 associated dementia (HAD) is not well understood. HIV-1 binds efficiently to astrocytes but infects only a small fraction of the cells in vitro and in vivo. To gain insight into the biology of HIV-1-expressing astrocytes, we productively infected human fetal astrocytes with pseudotyped HIV-1 and employed Affymetrix oligonucleotide microarrays to determine global changes in cellular gene expression at the peak of virus production. With a twofold change as a cutoff, HIV-1 increased transcription of 266 genes in astrocytes and suppressed expression of 468. The functions of highly expressed genes included interferon-mediated antiviral responses (OAS1, IFIT1), intercellular contacts (SH3, glia-derived nexin), cell homing/adhesion (matrix metalloproteinases), and cell-cell signaling (neuropilin 1 and 2). Surprisingly, genes involved in innate immune responses of astrocytes were largely unaffected. The single most significant effect of HIV-1, however, was down-modulation of at least 55 genes involved in control of cell cycle, DNA replication, and cell proliferation, which were overrepresented in these categories with probability scores of 10(-10)-10(-26). Our data suggest that HIV-1 expression in astrocytes profoundly alters host cell biology, with potential consequences for the physiological function of astrocytes during HIV-1 infection in the brain.

A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages.

A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages. PMID:24945246

X-ray microanalysis was used to study the patterns of K+, Na+ and Cl- accumulation in salinized (25 mm NaCl) and non-salinized grapevine (Vitis) roots. The aim was to determine whether NaCl affects patterns of Cl- accumulation differentially in the roots of a Cl--excluding genotype and a non-excluding genotype. Two regions of fibrous roots were analysed: (1) a region 2-3 mm basipetal to the root tip; and (2) a region of the root 10-12 mm basipetal to the root tip where the outermost layer is the hypodermis. The ion contents of the hypodermis, cortex, endodermis and pericycle vacuoles were analysed. Data were also collected from the cytoplasm of the endodermal and pericycle cells. The analyses showed that the ion profiles of the hypodermis and the endodermis were significantly different from those of the cortex and pericycle. The hypodermis and endodermis had higher K+ and lower Na+ and Cl- than surrounding cells. Some changes due to salinity such as increased K+ concentrations in the hypodermis were also noted. Chloride concentrations did not differ between the genotypes in the hypodermis, across the cortex or in the endodermis, but were higher in the pericycle of the excluder in comparison with the non-excluding genotype. However, K+/Na+ ratios of the cortex and endodermis were higher in the excluder. The pericycle cells exhibited the greatest ability to sequester Na+ and Cl- in vacuoles. Overall the data show cell-type-specific ion accumulation patterns and small but significant differences were found between genotypes. The possibility that these accumulation patterns arise from differences in uptake properties of cell types and/or result from the spatial distribution of the cell types along the competing symplastic and apoplastic ion transport pathways across the root is discussed.

Obesity and type 2 diabetes mellitus represent a significant global health crisis. These two interrelated diseases are typified by perturbed insulin signaling in the hypothalamus. Using novel hypothalamic cell lines, we have begun to elucidate the molecular and intracellular mechanisms involved in the hypothalamic control of energy homeostasis and insulin resistance. In this review, we present evidence of insulin and glucose signaling pathways that lead to changes in neuropeptide gene expression. We have identified some of the molecular mechanisms involved in the control of de novo hypothalamic insulin mRNA expression. And finally, we have defined key mechanisms involved in the etiology of cellular insulin resistance in hypothalamic neurons that may play a fundamental role in cases of high levels of insulin or saturated fatty acids, often linked to the exacerbation of obesity and diabetes.

Lidamycin (LDM) is a novel member of the enediyne antibiotics identified in China with potent antitumor activity. However, it remains unclear whether LDM has potential molecular targets that may affect its antitumor activity. Enhancer of zeste homolog 2 (EZH2) functions as a histone lysine methyltransferase and mediates trimethylation on histone 3 lysine 27 (H3K27me3). High EZH2 level is found to be positively correlated with the aggressiveness, metastasis and poor prognosis of cancer. Here, we aim to study the role of EZH2 in LDM-induced senescence, as well as in the cytotoxicity of LDM in human colon cancer cells. LDM is found to be relatively more potent in inhibiting the colon cancer cells harboring high EZH2 level and induces irreversible cellular senescence at IC50 dose range, as evidenced by senescence-associated β-galactosidase staining, cell cycle arrest and molecular changes of senescence regulators including p21 in HCT116 and SW620 cells. More importantly, LDM is found to markedly inhibit EZH2 expression at both protein and mRNA levels upon the induction of p21 and cellular senescence. LDM also selectively inhibits EZH2 expression as compared with other histone lysine methyltransferases. Knockdown of p21 with siRNAs abolishes LDM-induced senescence, whereas EZH2 knockdown markedly increases p21 expression and causes senescent phenotype. Enrichment of both EZH2 and H3K27me3 levels in the p21 promoter region is reduced by LDM. Moreover, EZH2 overexpression reduces cellular senescence, p21 expression and DNA damage response upon LDM exposure. LDM also demonstrates potent antitumor efficacy in xenografted animal models. Collectively, our work provides first demonstration that EZH2 may mediate, at least partially, the senescence-inducing effects of LDM by regulating p21 expression and DNA damage effect. Thus, EZH2 may serve as a potential target and biomarker to indicate the clinical efficacy of the potent enediyne antitumor drug. PMID:27882937

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a housekeeping gene product present in all cells, is an essential enzyme of catabolic glycolysis and anabolic gluconeogenesis, and regulates tumor cell growth and metastasis. Because glycolytic enzyme up-regulation of expression contributes to glycolytic flux, leading to increased of cell growth and a resistance to cellular stress of normal fibroblasts whereas down-regulation of PGI/AMF leads to mesenchymal-to-epithelial transition in tumor cells, we examined the involvement of PGI/AMF in overcoming cellular senescence in cancer cells. PGI/AMF cellularexpression in HT1080 human fibrosarcoma was down-regulated by small interfering RNA methodology, which resulted in an increased sensitivity to oxidative stress and oxidative stress-induced cellular senescence. Signaling analysis revealed that the senescence pathway involving p21 cyclin-dependent kinase inhibitor was up-regulated in PGI/AMF knockdown cells and that superoxide dismutase is the upstream regulator protein of p21-mediated cellular senescence. A specific inhibitor of PGI/AMF induced cellular senescence and p21 expression in tumor cells exposed to an oxidative stress environment. Taken together, the results presented here suggest that PGI/AMF is involved in oxidative stress-induced cellular senescence and should bring novel insights into the control of cellular growth leading to a new methodology for cancer treatment.

Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9) technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO) cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties. PMID:27304954

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response.

Phospholipid signaling has been recently implicated in diverse cellular processes in higher plants. We identified a cDNA encoding the phospholipase A1 homolog (CaPLA1) from 5-day-old early roots of hot pepper. The deduced amino acid sequence showed that the lipase-specific catalytic triad is well conserved in CaPLA1. In vitro lipase assays and site-directed mutagenesis revealed that CaPLA1 possesses PLA1 activity, which catalyzes the hydrolysis of phospholipids at the sn-1 position. CaPLA1 was selectively expressed in young roots, at days 4-5 after germination, and rapidly declined thereafter, suggesting that the expression of CaPLA1 is subject to control by a development-specific mechanism in roots. Because transgenic work was extremely difficult in hot peppers, in this study we overexpressed CaPLA1 in Arabidopsis so as to provide cellular information on the function of this gene. CaPLA1 overexpressors had significantly longer roots, leaves and petioles, and grew more rapidly than the wild-type plants, leading to an early bolting phenotype with prolonged inflorescence. Microscopic analysis showed that the vegetative tissues of 35S:CaPLA1 plants contained an increased number of small-sized cells, which resulted in highly populated cell layers. In addition, mRNAs for cell cycle-controlled proteins and fatty acid catabolizing enzymes were coordinately upregulated in CaPLA1-overexpressing plants. These results suggest that CaPLA1 is functionally relevant in heterologous Arabidopsis cells, and hence might participate in a subset of positive control mechanisms of cell and tissue growth in transgenic lines. We discuss possible biochemical and cellular functions of CaPLA1 in relation to the phospholipid signaling pathway in hot pepper and transgenic Arabidopsis plants.

It is well known that the polyamines spermidine and spermine, along with the diamine putrescine, are involved in many cellular processes and they are known to play an important role in the control of the innate immune response in higher vertebrates. However, to the best of our knowledge, no studies have focused on their immunological implications in other vertebrates, such as fish. For this reason, the effects of polyamines on the cellular innate immune response and immune-related gene expression were evaluated in vitro, using seabream head-kidney leucocytes (HKL). For this study, head-kidney leucocytes were incubated with the polyamines putrescine, spermine or spermidine (0.005 and 0.0025%) for 0.50, 1, 2 or 4 h. No significant effect was observed on either leucocyte viability or the innate cellular immune responses (peroxidase content and phagocytic and respiratory burst activities). The polyamines produced an increase in respiratory burst and phagocytic ability when leucocytes were incubated principally with putrescine (0.005 and 0.0025%) after 2 and 4 h of the experiment. Finally, the expression levels of immune-associated genes (IgM, MHCIα, MHCIIα, C3, IL-1β, CD8, Hep, NCCRP-1, CSF-1 and TLR) were quantified by real-time PCR and some of them (C3, MHCI, CD8, IgM and Hep) were up-regulated by the higher polyamine concentration. Further studies are needed to ascertain how polyamines control the immune system of seabream as well as which mechanisms are involved.

Transcription of HIV-1 genes depends on the RNA polymerase II kinase and elongation factor positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We identify histone H1 as a substrate for P-TEFb involved in cellular and HIV-1 transcription. We show that P-TEFb interacts with H1 and that P-TEFb inhibition by RNAi, flavopiridol, or dominant negative CDK9 expression correlates with loss of phosphorylation and mobility of H1 in vivo. Importantly, P-TEFb directs H1 phosphorylation in response to wild-type HIV-1 infection, but not Tat-mutant HIV-1 infection. Our results show that P-TEFb phosphorylates histone H1 at a specific C-terminal phosphorylation site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb also disrupts Tat transactivation in an HIV reporter cell line as well as transcription of the c-fos and hsp70 genes in HeLa cells. We identify histone H1 as a novel P-TEFb substrate, and our results suggest new roles for P-TEFb in both cellular and HIV-1 transcription. PMID:20551309

From in vivo single-event measurements of the transient and steady-state transcription activity of a single-copy lac-ara-1 promoter in Escherichia coli, we characterize the intake kinetics of its inducer (IPTG) from the media. We show that the empirical data are well-fit by a model of intake assuming a bilayer membrane, with the passage through the second layer being rate-limiting, coupled to a stochastic, sub-Poissonian, multi-step transcription process. Using this model, we show that for a wide range of extracellular inducer levels (up to 1.25 mM) the intake process is diffusive-like, suggesting unsaturated membrane permeability. Inducer molecules travel from the periplasm to the cytoplasm in, on average, 31.7 minutes, strongly affecting cells' response time. The novel methodology followed here should aid the study of cellular intake mechanisms at the single-event level.

Background: Processing of microRNAs (miRNAs) is a highly controlled process. Deregulation of miRNA expression was observed in several types of cancer but changes in the miRNA-processing enzymes have not been analysed until today. In this study, we analysed Argonaute2 (AGO2, EIF2C2), as one main factor of the miRNA processing ensemble, in the context of cancer development, especially in melanoma. Methods: We determined the AGO2 expression level in melanoma, as well as in other cancers, with biochemical approaches (qRT–PCR, western blot and immunofluorescence studies) and analysed the cell behaviour in migration assays. Results: Specifically in melanoma, we revealed a strong reduction of AGO2 expression compared with primary melanocytes. The reduction of AGO2 expression was only found on protein level, whereas the mRNA level stayed unchanged hinting to post-transcriptional regulation. We could show that re-expression of AGO2 in melanoma leads to a strong improvement of regulatory effects due to increased functionality of small-interfering RNAs and short hairpin RNAs. Conclusion: We identified melanoma-specific downregulation of AGO2 and corresponding reduced RNAi efficiency. These findings will help to understand the molecular basis of malignant melanoma and can potentially lead to an improvement of therapeutic strategies. PMID:24169347

The present study aimed to describe and characterize the cellular components during the evolution of chronic granulomatous inflammation in the teleost fish pacus (P. mesopotamicus) induced by Bacillus Calmette-Guerin (BCG), using S-100, iNOS and cytokeratin antibodies. 50 fish (120±5.0 g) were anesthetized and 45 inoculated with 20 μL (40 mg/mL) (2.0 x 106 CFU/mg) and five inoculated with saline (0,65%) into muscle tissue in the laterodorsal region. To evaluate the inflammatory process, nine fish inoculated with BCG and one control were sampled in five periods: 3rd, 7th, 14th, 21st and 33rd days post-inoculation (DPI). Immunohistochemical examination showed that the marking with anti-S-100 protein and anti-iNOS antibodies was weak, with a diffuse pattern, between the third and seventh DPI. From the 14th to the 33rd day, the marking became stronger and marked the cytoplasm of the macrophages. Positivity for cytokeratin was initially observed in the 14th DPI, and the stronger immunostaining in the 33rd day, period in which the epithelioid cells were more evident and the granuloma was fully formed. Also after the 14th day, a certain degree of cellular organization was observed, due to the arrangement of the macrophages around the inoculated material, with little evidence of edema. The arrangement of the macrophages around the inoculum, the fibroblasts, the lymphocytes and, in most cases, the presence of melanomacrophages formed the granuloma and kept the inoculum isolated in the 33rd DPI. The present study suggested that the granulomatous experimental model using teleost fish P. mesopotamicus presented a similar response to those observed in mammals, confirming its importance for studies of chronic inflammatory reaction. PMID:25811875

Objective: The objective of this study was to provide insight into the molecular mechanisms of acute ischemic cerebrovascular syndrome (AICS) through gene expression profiling and pathway analysis. Methods: Peripheral whole blood samples were collected from 39 MRI-diagnosed patients with AICS and 25 nonstroke control subjects ≥18 years of age. Total RNA was extracted from whole blood stabilized in Paxgene RNA tubes, amplified, and hybridized to Illumina HumanRef-8v2 bead chips. Gene expression was compared in a univariate manner between stroke patients and control subjects using t test in GeneSpring. The significant genes were tested in a logistic regression model controlling for age, hypertension, and dyslipidemia. Inflation of type 1 error was corrected by Bonferroni and Ingenuity Systems Pathway analysis was performed. Validation was performed by QRT-PCR using Taqman gene expression assays. Results: A 9-gene profile was identified in the whole blood of ischemic stroke patients using gene expression profiling. Five of these 9 genes were identified in a previously published expression profiling study of stroke and are therefore likely biomarkers of stroke. Pathway analysis revealed toll-like receptor signaling as a highly significant canonical pathway present in the peripheral whole blood of patients with AICS. Conclusions: Our study highlights the relevance of the innate immune system through toll-like receptor signaling as a mediator of response to ischemic stroke and supports the claim that gene expression profiling can be used to identify biomarkers of ischemic stroke. Further studies are needed to validate and refine these biomarkers for their diagnostic potential. GLOSSARY AICS = acute ischemic cerebrovascular syndrome; BBB = blood–brain barrier; IPA = Ingenuity Systems Pathway analysis; PBMC = peripheral blood mononuclear cell; rtPA = recombinant tissue plasminogen activator; TLR = toll-like receptor. PMID:20837969

Purpose A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores, the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity. Materials and Methods Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632. Results When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation induced double strand break (DSB) repair, systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1. Conclusion CFL-1 over-expression enhances radiosensitivity and this

Immunoreactivity for both processed and unprocessed forms of chromogranin A (CGA) was examined, using an antibody recognizing the WE14 epitope, among terminal fields and cell bodies of anatomically defined GABAergic, glutamatergic, cholinergic, catecholaminergic, and peptidergic cell groups in the rodent central nervous system. CGA is ubiquitous within neuronal cell bodies, with no obvious anatomical or chemically-coded subdivision of the nervous system in which CGA is not expressed in most neurons. CGA expression is essentially absent from catecholaminergic terminal fields in the CNS, suggesting a relative paucity of large dense-core vesicles in CNS compared to peripheral catecholaminergic neurons. Extensive synaptic co-localization with classical transmitter markers is not observed even in areas such as amygdala, where CGA fibers are numerous, suggesting preferential segregation of CGA to peptidergic terminals in CNS. Localization of CGA in dendrites in some areas of CNS may indicate its involvement in regulation of dendritic release mechanisms. Finally, the ubiquitous presence of CGA in neuronal cell somata, especially pronounced in GABAergic neurons, suggests a second non-secretory vesicle-associated function for CGA in CNS. We propose that CGA may function in the CNS as a prohormone and granulogenic factor in some terminal fields, but also possesses as-yet unknown unique cellular functions within neuronal somata and dendrites. PMID:20005907

The placenta expressed transcript 1 (PLET1) gene, which is expressed in placentas of pigs and mice, has been found to have a potential role in trophoblast cell fate decision in mice. Results of this study showed that the porcine PLET1 mRNA and protein were expressed exclusively in trophoblast cells on Days 15, 26, 50, and 95 of gestation (gestation length in the pig is 114 days), indicating that the PLET1 could be a useful marker for porcine trophoblast cells. Additionally, PLET1 protein was found to be redistributed from cytoplasm to the apical side of trophoblast cells as gestation progresses, which suggests a role of PLET1 in the establishment of a stable trophoblast and endometrial epithelial layers. In addition, two transcripts that differ in the 3′ UTR length but encode identical protein were identified to be generated by the alternative cleavage and polyadenylation (APA), and the expression of PLET1-L transcript was significantly upregulated in porcine placentas as gestation progresses. Furthermore, we demonstrated the interaction between the miR-365-3p and PLET1 gene using luciferase assay system. Our findings imply an important role of PLET1 in the placental development in pigs. PMID:27941613

The placenta expressed transcript 1 (PLET1) gene, which is expressed in placentas of pigs and mice, has been found to have a potential role in trophoblast cell fate decision in mice. Results of this study showed that the porcine PLET1 mRNA and protein were expressed exclusively in trophoblast cells on Days 15, 26, 50, and 95 of gestation (gestation length in the pig is 114 days), indicating that the PLET1 could be a useful marker for porcine trophoblast cells. Additionally, PLET1 protein was found to be redistributed from cytoplasm to the apical side of trophoblast cells as gestation progresses, which suggests a role of PLET1 in the establishment of a stable trophoblast and endometrial epithelial layers. In addition, two transcripts that differ in the 3' UTR length but encode identical protein were identified to be generated by the alternative cleavage and polyadenylation (APA), and the expression of PLET1-L transcript was significantly upregulated in porcine placentas as gestation progresses. Furthermore, we demonstrated the interaction between the miR-365-3p and PLET1 gene using luciferase assay system. Our findings imply an important role of PLET1 in the placental development in pigs.

ABSTRACT High-risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). The production of multiple mRNA isoforms from a single gene is controlled by serine/arginine-rich splicing factors (SRSFs), and HPV16 infection induces overexpression of a subset of these, SRSFs 1, 2, and 3. In this study, we examined whether these proteins could control HPV16 oncoprotein expression. Small interfering RNA (siRNA) depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression, depletion of SRSF2 resulted in a significant loss of E6E7 RNAs, resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumor phenotype of HPV16-positive cervical cancer cells, as its depletion resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis. SRSF2 did not affect transcription from the P97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumor maintenance. IMPORTANCE Expression of the HPV16 oncoproteins E7 and E6 drives HPV-associated tumor formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that the RNAs can have increased stability upon integration into the host genome. SR splicing factors (SRSFs) control splicing but can also control other events in the RNA life cycle, including RNA stability. Previously, we demonstrated increased levels of SRSFs 1, 2, and 3 during

Summary We investigated miRNA expression changes associated with stress-induced premature senescence (SIPS) in primary cultures of human diploid fibroblasts (HDF) and human trabecular meshwork (HTM) cells. Twenty-five miRNAs were identified by miRNA microarray analysis and their changes in expression were validated by TaqMan realtime RT-PCR in three independent cell lines of HTM and HDF. SIPS in both HTM and HDF cell types was associated with significant down-regulation of four members of the miR-15 family and five miRNAs of the miR-106b family located in the oncogenic clusters miR-17–92, miR-106a-363, and miR-106b-25. SIPS was also associated with up-regulation of two miRNAs (182 and 183) from the miR-183-96-182 cluster. Transfection with miR-106a agomir inhibited the up-regulation of p21CDKN1A associated with SIPS while transfection with miR-106a antagomir led to increased p21CDKN1A expression in senescent cells. In addition, we identified retinoic acid receptor gamma (RARG) as a target of miR-182 and showed that this protein was down-regulated during SIPS in HDF and HTM cells. These results suggest that changes in miRNA expression might contribute to phenotypic alterations of senescent cells by modulating the expression of key regulatory proteins such as p21CDKN1A as well as by targeting genes that are down-regulated in senescent cells such as RARG. PMID:19782699

The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or {gamma}-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to {sup 60}Co {gamma} rays or 254-m UV radiation. Differential display of cDNAs and Northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a ``crisis`` period was evident during which time cell growth in high serum (20%) was no longer optimal, and serum concentrations were reduced (to 10%) to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant- than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of {gamma}-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following {gamma}-ray exposure of the intermediate (passage 45) epithelial cells. Differential display, however, revealed changes in expression of several transcripts following exposure to ionizing and ultraviolet radiations. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. We are conducting experiments to identify these genes.

The ability to cope with high temperature variations is a critical factor in intertidal communities. Two species of intertidal rocky shore shrimps (Palaemon sp.) with different vertical distributions were collected from the Portuguese coast in order to test if they were differentially sensitive to thermal stress. Three distinct levels of biological organization (organismal, biochemical, and cellular) were surveyed. The shrimp were exposed to a constant rate of temperature increase of 1°C x h(-1), starting at 20°C until reaching the CTMax (critical thermal maximum). During heat stress, two biomarkers of protein damage were quantified in the muscle via enzyme-linked immunosorbent assays: heat shock proteins HSP70 (hsp70/hsc70) and total ubiquitin. Muscle histopathological alterations caused by temperature were also evaluated. CTMax values were not significantly different between the congeners (P. elegans 33.4 ± 0.5 °C; P. serratus 33.0 ± 0.5 °C). Biomarker levels did not increase along the temperature trial, but P. elegans (higher intertidal) showed higher amounts of HSP70 and total ubiquitin than P. serratus (lower intertidal). HSP70 and total ubiquitin levels showed a positive significant correlation in both species, suggesting that their association is important in thermal tolerance. Histopathological observations of muscle tissue in P. serratus showed no gross alterations due to temperature but did show localized atrophy of muscle fibers at CTMax. In P. elegans, alterations occurred at a larger scale, showing multiple foci of atrophic muscular fascicles caused by necrotic or autolytic processes. In conclusion, Palaemon congeners displayed different responses to stress at a cellular level, with P. elegans having greater biomarker levels and histopathological alterations.

Bacterial self-replication is a complex process composed of many de novo synthesis steps catalyzed by a myriad of molecular processing units, e.g., the transcription-translation machinery, metabolic enzymes, and the replisome. Successful completion of all production tasks requires a schedule-a temporal assignment of each of the production tasks to its respective processing units that respects ordering and resource constraints. Most intracellular growth processes are well characterized. However, the manner in which they are coordinated under the control of a scheduling policy is not well understood. When fast replication is favored, a schedule that minimizes the completion time is desirable. However, if resources are scarce, it is typically computationally hard to find such a schedule, in the worst case. Here, we show that optimal scheduling naturally emerges in cellular self-replication. Optimal doubling time is obtained by maintaining a sufficiently large inventory of intermediate metabolites and processing units required for self-replication and additionally requiring that these processing units be "greedy," i.e., not idle if they can perform a production task. We calculate the distribution of doubling times of such optimally scheduled self-replicating factories, and find it has a universal form-log-Frechet, not sensitive to many microscopic details. Analyzing two recent datasets of Escherichia coli growing in a stationary medium, we find excellent agreement between the observed doubling-time distribution and the predicted universal distribution, suggesting E. coli is optimally scheduling its replication. Greedy scheduling appears as a simple generic route to optimal scheduling when speed is the optimization criterion. Other criteria such as efficiency require more elaborate scheduling policies and tighter regulation.

Recent advances in sequencing technologies have revealed that the genome is extensively transcribed, yielding a large repertoire of noncoding RNAs. These include long noncoding RNAs (lncRNAs), mRNA-like molecules that do not code for proteins, which are emerging as a new class of RNAs that play important roles in a variety of cellular processes. Ongoing studies are revealing new insights about lncRNAs, including their physiological functions, disease relationships, and molecular mechanisms of action. Characterized lncRNAs have been shown to interact with and modulate the activity of other RNAs and protein partners, leading to alterations in transcriptional and posttranscriptional regulatory processes. In this review, we summarize the key features of lncRNAs, their molecular mechanisms of action, biological functions, and therapeutic implications, particularly as they apply to the field of molecular endocrinology. In addition, we provide a brief overview of how molecular biologists are beginning to probe the identity, mechanisms, and functions of this emerging class of RNA molecules. PMID:23885095

The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays a complex role during the replication of primate lentiviruses. On the one hand, NF-κB is essential for induction of efficient proviral gene expression. On the other hand, this transcription factor contributes to the innate immune response and induces expression of numerous cellular antiviral genes. Recent data suggest that primate lentiviruses cope with this challenge by boosting NF-κB activity early during the replication cycle to initiate Tat-driven viral transcription and suppressing it at later stages to minimize antiviral gene expression. Human and simian immunodeficiency viruses (HIV and SIV, respectively) initially exploit their accessory Nef protein to increase the responsiveness of infected CD4+ T cells to stimulation. Increased NF-κB activity initiates Tat expression and productive replication. These events happen quickly after infection since Nef is rapidly expressed at high levels. Later during infection, Nef proteins of HIV-2 and most SIVs exert a very different effect: by down-modulating the CD3 receptor, an essential factor for T cell receptor (TCR) signaling, they prevent stimulation of CD4+ T cells via antigen-presenting cells and hence suppress further induction of NF-κB and an effective antiviral immune response. Efficient LTR-driven viral transcription is maintained because it is largely independent of NF-κB in the presence of Tat. In contrast, human immunodeficiency virus type 1 (HIV-1) and its simian precursors have lost the CD3 down-modulation function of Nef and use the late viral protein U (Vpu) to inhibit NF-κB activity by suppressing its nuclear translocation. In this review, we discuss how HIV-1 and other primate lentiviruses might balance viral and antiviral gene expression through a tight temporal regulation of NF-κB activity throughout their replication cycle. PMID:28261165

The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays a complex role during the replication of primate lentiviruses. On the one hand, NF-κB is essential for induction of efficient proviral gene expression. On the other hand, this transcription factor contributes to the innate immune response and induces expression of numerous cellular antiviral genes. Recent data suggest that primate lentiviruses cope with this challenge by boosting NF-κB activity early during the replication cycle to initiate Tat-driven viral transcription and suppressing it at later stages to minimize antiviral gene expression. Human and simian immunodeficiency viruses (HIV and SIV, respectively) initially exploit their accessory Nef protein to increase the responsiveness of infected CD4(+) T cells to stimulation. Increased NF-κB activity initiates Tat expression and productive replication. These events happen quickly after infection since Nef is rapidly expressed at high levels. Later during infection, Nef proteins of HIV-2 and most SIVs exert a very different effect: by down-modulating the CD3 receptor, an essential factor for T cell receptor (TCR) signaling, they prevent stimulation of CD4(+) T cells via antigen-presenting cells and hence suppress further induction of NF-κB and an effective antiviral immune response. Efficient LTR-driven viral transcription is maintained because it is largely independent of NF-κB in the presence of Tat. In contrast, human immunodeficiency virus type 1 (HIV-1) and its simian precursors have lost the CD3 down-modulation function of Nef and use the late viral protein U (Vpu) to inhibit NF-κB activity by suppressing its nuclear translocation. In this review, we discuss how HIV-1 and other primate lentiviruses might balance viral and antiviral gene expression through a tight temporal regulation of NF-κB activity throughout their replication cycle.

Human cells express four mitochondrial adenine nucleotide translocase (hANT) isoforms that are tissue-specific and developmentally regulated. hANT1 is mainly expressed in terminally differentiated muscle cells; hANT2 is growth-regulated and is upregulated in highly glycolytic and proliferative cells; and hANT3 is considered to be ubiquitous and non-specifically regulated. Here, we studied how the expression of hANT isoforms is regulated by proliferation and in response to metabolic stimuli, and examined the metabolic consequences of their silencing and overexpression. In HeLa and HepG2 cells, expression of hANT3 was upregulated by shifting metabolism towards oxidation or by slowed growth associated with contact inhibition or growth-factor deprivation, indicating that hANT3 expression is highly regulated. Under these conditions, changes in hANT2 mRNA expression were not observed in either HeLa or HepG2 cells, whereas in SGBS preadipocytes (which, unlike HeLa and HepG2 cells, are growth-arrest-sensitive cells), hANT2 mRNA levels decreased. Additionally, overexpression of hANT2 promoted cell growth and glycolysis, whereas silencing of hANT3 decreased cellular ATP levels, limited cell growth and induced a stress-like response. Thus, cancer cells require both hANT2 and hANT3, depending on their proliferation status: hANT2 when proliferation rates are high, and hANT3 when proliferation slows. PMID:26842067

In this study the expression of a regulatory cross-reactive idiotype (SJ-CRIM), which is associated with anti-soluble egg antigen (SEA) molecules in murine Schistosoma japonicum infection, is described. Both humoral and cellular components of the immune response were analyzed during the course of infection with S. japonicum. In the humoral immune response, the content of SJ-CRIM decreases as the titer of anti-SEA antibody increases throughout infection. Quantitatively, values for serum ranged from 13.8 +/- 0.3 micrograms of SJ-CRIM, which binds anti-idiotypic antibody per ml of serum at 6 weeks postinfection, to 1.3 +/- 1.8 micrograms/ml at 30 weeks postinfection. Analysis of splenic cell subpopulations for expression of SJ-CRIM revealed that only splenic B cells expressed SJ-CRIM during acute infection (5 to 10 weeks postinfection). On the other hand, thymic cells with a high expression of the SJ-CRIM and Ly-1 marker were observed in acute infections up to 15 weeks postinfection. These data indicate that SJ-CRIM-bearing T cells are selectively localized in acute infection. In addition, the disappearance of expression of SJ-CRIM in serum and cells of chronically infected animals parallels the modulation of granulomatous inflammation and portal hypertension. Results of this study suggest that expression of SJ-CRIM on anti-SEA molecules could represent a marker for acute infection, while its disappearance from serum serves as a marker for modulation of disease. PMID:2873105

Filamentous fungi are often used as cell factories for recombinant protein production because of their ability to secrete large quantities of hydrolytic enzymes. However, even using strong transcriptional promoters, yields of nonfungal proteins are generally much lower than those of fungal proteins. Recent analyses revealed that expression of certain nonfungal secretory proteins induced the unfolded protein response (UPR), suggesting that they are recognized as proteins with folding defects in filamentous fungi. More recently, however, even highly expressed endogenous secretory proteins were found to evoke the UPR. These findings raise the question of whether the unfolded or misfolded state of proteins is selectively recognized by quality control mechanisms in filamentous fungi. In this study, a fungal secretory protein (1,2-α-D-mannosidase; MsdS) with a mutation that decreases its thermostability was expressed at different levels in Aspergillus oryzae. We found that, at moderate expression levels, wild-type MsdS was secreted to the medium, while the mutant was not. In the strain with a deletion for the hrdA gene, which is involved in the endoplasmic reticulum-associated degradation pathway, mutant MsdS had specifically increased levels in the intracellular fraction but was not secreted. When overexpressed, the mutant protein was secreted to the medium to a similar extent as the wild-type protein; however, the mutant underwent hyperglycosylation and induced the UPR. Deletion of α-amylase (the most abundant secretory protein in A. oryzae) alleviated the UPR induction by mutant MsdS overexpression. These findings suggest that misfolded MsdS and unfolded species of α-amylase might act synergistically for UPR induction.

Heat stress is an important domain of research in livestock due to its negative impact on production and disease resistance. The augmentation of stress in the body stimulates the antioxidative activity comprising various enzymes (viz., catalase, superoxide dismutase), metabolites (reduced glutathione, etc.), vitamins, minerals, etc. to combat the situation. The major key players involved in regulation of heat shock response in eukaryotes are the transcription factors, called as heat shock factors (HSF). They activate the heat shock protein (HSP) genes by binding to their promoters. Lymphocytes are considered to be the best model to evaluate the immunity in any living body as it contains plethora of white blood cells (WBCs).In this study, the peripheral blood mononuclear cells (PBMC) obtained from non-lactating Sahiwal vis-à-vis crossbred (Holstein Friesian × Sahiwal) cattle with 75% or more exotic inheritance were subjected to heat shock at 39, 41, and 43 °C in three different incubators, in vitro. The cell count and viability test of pre and post heat stress of concerned PBMCs indicated that the crossbreeds are more prone to heat stress as compared to Sahiwal. The reverse transcription PCR (qRT-PCR) expression data revealed an increment in HSF1 expression at 41 °C which subsequently declined (non-significantly) at 43 °C in both breeds post 1 h heat shock. However, the association between the HSF 1 expression and antioxidative activity through correlation analysis was found to be non-significant (P < 0.05), though enzymatic activity appeared to behave in a similar fashion in both breeds at 5% level of significance (P < 0.05). This rule out the role of HSF1 expression level on the activity of enzymes involved in oxidative stress in vitro in zebu and crossbred cattle.

The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS. PMID:12736376

Sodium methyldithiocarbamate (SMD) is one of the most abundantly used conventional pesticides in the United States. At dosages relevant to occupational exposure, it causes major effects on the immune system in mice, including a decreased resistance to sepsis. This lab has identified some of the mechanisms of action of this compound and some of the immunological parameters affected, but the global effects have not previously been assessed. The purpose of the present study was to conduct transcriptomic analysis of the effects of SMD on lipopolysaccharide-induced expression of mediators important in innate immunity and inflammation. The results revealed broad effects on expression of transcription factors in both branches of Toll-like receptor 4 (TLR4) signaling (MyD88 and TRIF). However, TLR3 and interferon signaling pathways were decreased to a greater extent, and assessment of the effects of SMD on polyinosinic polycytidylic acid–induced cytokine and chemokine production revealed that these responses mediated by TLR3 were indeed sensitive to the effects of SMD, with inhibition occurring at lower dosages than required to inhibit responses to other immunological stimuli tested in our previous studies. In the downstream signaling pathways of these TLRs, functional analysis also revealed that NF-κB activation was inhibited by SMD, as indicated by gene expression analysis and a reporter construct in mice. A previously unreported effect on luteinizing hormone and follicle-stimulating hormone pathways was also observed. PMID:24056979

It has long been held that the cold-blooded vertebrates lack mammalian-like germinal centers, though they do have affinity maturation and the immunoglobulin mutator activation-induced cytidine deaminase or AID. Using AID as a marker of sites of somatic hypermutation, we have identified discrete cell clusters of up to several thousand cells, in the spleen and kidney of channel catfish (Ictalurus punctatus), which may be primordial germinal centers. In situ hybridization revealed that AID expressing cells are interspersed or surrounded by a population of pigmented CSF1-R expressing cells called melano-macrophages. Significantly, melano-macrophages or associated reticular cells have been previously noted for their ability to retain soluble antigen on or near their surface for several weeks following vaccination. Laser capture microdissection and RT-PCR were used to establish that these cell clusters also contained cells expressing Ig heavy chain transcripts as well as transcripts of TcRbeta and the putative CD4 homologue of fish. These observations, coupled with past work showing that mutations develop in B-cell lineages in fishes, allow us to develop a model for how affinity maturation may have evolved in early gnathostome vertebrates.

The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. The function of thapsigargin-sensitive but not thapsigargin-resistant (Ca2+-Mg2+)ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca2+ uptake experiments, was significantly depressed by 30-50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase type 2 protein expression, without any significant change in plasma membrane (Ca2+-Mg2+)ATPase expression. Finally, [3H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. The present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens.

Past studies have shown that both hypo- and hyper-gravity have significant consequences on a variety of tissues and organ systems. It is not known if the effects of environmental stimuli such as altered gravity are beneficial or detrimental, and if the effects can be prevented or reversed. Animal experiments from the Space Lab and Cosmos missions indicate that events that are mediated by cyclic AMP, such as cellular responses to catecholamine and peptide hormone action, are significantly altered in a number of tissues as a consequence of space flight. A secretory cyclic AMP-receptor protein (cARP), is present in saliva, and can serve as an indicator of individual responses to physiologic and environmental stress. Animal experiments have shown that the hypergravity component of space flight is a significant stress factor. In humans, cARP levels in each individual are constant under normal conditions, but elevated after acute stress. Additionally, the levels of cARP in secreted saliva can be compared to those in gingival crevicular fluid (GCF), which reflects the protein composition of serum. The ratio of cARP in saliva to that in GCF can be used as a measure of basal compared to hyper-or hypo-gravity values. An ultimate goal is to test hyper and zero G responses in human saliva to determine if cARP is a suitable index of acute and chronic stress. A miniaturized test kit for saliva collection has been designed. Samples can be collected and stored till analyses are carried out that will distinguish the effects of increased gravity from those of one and zero G. Such tests can serve as an individualized monitoring system for physiologic responses either in space or on earth. .

One method of iron transport across the blood brain barrier (BBB) involves the transferrin receptor (TR), which is localized to the specialized brain capillary endothelium. The melanotransferrin (MTf) molecule, also called p97, has been widely described as a melanoma specific molecule, however, its expression in brain tissues has not been addressed. MTf has a high level of sequence homology to transferrin (Tf) and lactoferrin, but is unusual because it predominantly occurs as a membrane bound, glycosylphosphatidylinositol (GPI) anchored molecule, but can also occur as a soluble form. We have recently demonstrated that GPI-anchored MTf provides a novel route for cellular iron uptake which is independent of Tf and its receptor. Here we consider whether MTf may have a role in the transport of iron across the BBB. The distributions of MTf, Tf and the TR were studied immunohistochemically in human brain tissues. The distributions of MTf and TR were remarkably similar, and quite different from that of Tf. In all brain tissues examined, MTf and the TR were highly localized to capillary endothelium, while Tf itself was mainly localized to glial cells. These data suggest that MTf may play a role in iron transport within the human brain.

Spinal cord injury (SCI) is a severe traumatic disease in the central nervous system with high incidence and high morbidity. Recent study demonstrated that cell transplantation therapy may improve local microenvironment of the injury site and promote nerve regeneration to restore spinal cord functions. In this study, we constructed a glucocorticoid-induced bicistronic eukaryotic expression vector pGC-BDNF-IRES-NT3 by using molecular cloning techniques and examined the protective effect of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) expressed by this vector in a rat spinal cord injury (SCI) model. We first connected glucocorticoid response element (GRE) to cytomegalovirus (CMV) promoter and then the GRE-CMV gene was inserted into pEGFP-1 vector to construct the eukaryotic expression vector pGC-EGFP. Western blot analysis was used to confirm the expression of EGFP by transfecting this vector in RN-DSC cells. The IRES was used to connect BDNF gene and NT-3 gene and replaced the EGFP gene in pGC-EGFP plasmid to form the bicistronic expression vector-pGC-BDNF-IRES-NT3. After RN-DSC cells were transfected with the plasmid and treated with glucocorticoid, BDNF and NT-3 expression in the culture medium were measured by ELISA method. Finally, we found that combination therapy with the transfection of this vector and glucocorticoid had an anti-apoptotic effect in a cellular SCI model of RN-DSC cells. Therefore, the co-expression of BDNF and NT-3 by using this vector rescued the injured cells. This provided useful information for the gene-modification cell transplantation combined with glucocorticoid for the treatment of SCI.

Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2°C and +6°C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (±1°C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2°C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6°C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms. PMID:19777376

ABSTRACT The SaeRS two-component system is a master activator of virulence factor transcription in Staphylococcus aureus, but the cellular factors that control its activity are unknown. Fatty acid (FA) kinase is a two-component enzyme system required for extracellular FA uptake and SaeRS activity. Here, we demonstrate the existence of an intracellular nonesterified FA pool in S. aureus that is elevated in strains lacking FA kinase activity. SaeRS-mediated transcription is restored in FA kinase-negative strains when the intracellular FA pool is reduced either by growth with FA-depleted bovine serum albumin to extract the FA into the medium or by the heterologous expression of Neisseria gonorrhoeae acyl-acyl carrier protein synthetase to activate FA for phospholipid synthesis. These data show that FAs act as negative regulators of SaeRS signaling, and FA kinase activates SaeRS-dependent virulence factor production by lowering inhibitory FA levels. Thus, FA kinase plays a role in cellular lipid homeostasis by activating FA for incorporation into phospholipid, and it indirectly regulates SaeRS signaling by maintaining a low intracellular FA pool. PMID:28765222

Research on toxin-antitoxin loci (TA loci) is gaining impetus due to their ubiquitous presence in bacterial genomes and their observed roles in stress survival, persistence and drug tolerance. The present study investigates the expression profile of all the seventy-nine TA loci found in Mycobacterium tuberculosis. The bacterium was subjected to multiple stress conditions to identify key players of cellular stress response and elucidate a TA-coexpression network. This study provides direct experimental evidence for transcriptional activation of each of the seventy-nine TA loci following mycobacterial exposure to growth-limiting environments clearly establishing TA loci as stress-responsive modules in M. tuberculosis. TA locus activation was found to be stress-specific with multiple loci activated in a duration-based response to a particular stress. Conditions resulting in arrest of cellular translation led to greater up-regulation of TA genes suggesting that TA loci have a primary role in arresting translation in the cell. Our study identifed higBA2 and vapBC46 as key loci that were activated in all the conditions tested. Besides, relBE1, higBA3, vapBC35, vapBC22 and higBA1 were also upregulated in multpile stresses. Certain TA modules exhibited co-activation across multiple conditions suggestive of a common regulatory mechanism.

High voltage activated calcium channels are hetero-oligomeric protein complexes that mediate multiple cellular processes including the influx of extracellular Ca2+, neurotransmitter release, gene transcription and synaptic plasticity. These channels consist of a primary α1 pore-forming subunit, which is associated with an extracellular α2δ subunit and an intracellular β auxiliary subunit, which alter the gating properties and trafficking of the calcium channel. The cellular localization of the α2δ3 subunit in the mouse and rat retina is unknown. In this study, using RT-PCR a single band at ~305 bp corresponding to the predicted size of the α2δ3 subunit fragment was in mouse and rat retina and brain homogenates. Western blotting of rodent retina and brain homogenates showed a single 123 kDa band. Immunohistochemistry using an affinity purified antibody to the α2δ3 subunit revealed immunoreactive cell bodies in the ganglion cell layer (GCL) and inner nuclear layer (INL), and immunoreactive processes in the inner plexiform layer (IPL) and the outer plexiform layer (OPL). α2δ3 immunoreactivity was localized to multiple cell types, including ganglion, amacrine and bipolar cells, and photoreceptors, but not by horizontal cells. The expression of the α2δ3 calcium channel subunit to multiple cell types suggests this subunit participates widely in Ca channel-mediated signaling in the retina. PMID:25631988

Neuroblastoma, the major cause of infant cancer deaths, results from fast proliferation of undifferentiated neuroblasts. Treatment of high-risk neuroblastoma includes differentiation with retinoic acid (RA); however, the resistance of many of these tumors to RA-induced differentiation poses a considerable challenge. Human achaete-scute homolog 1 (hASH1) is a proneural basic helix-loop-helix transcription factor essential for neurogenesis and is often upregulated in neuroblastoma. Here, we identified a novel function for hASH1 in regulating the differentiation phenotype of neuroblastoma cells. Global analysis of 986 human neuroblastoma datasets revealed a negative correlation between hASH1 and neuron differentiation that was independent of the N-myc (MYCN) oncogene. Using RA to induce neuron differentiation in two neuroblastoma cell lines displaying high and low levels of hASH1 expression, we confirmed the link between hASH1 expression and the differentiation defective phenotype, which was reversed by silencing hASH1 or by hypoxic preconditioning. We further show that hASH1 suppresses neuronal differentiation by inhibiting transcription at the RA receptor element. Collectively, our data indicate hASH1 to be key for understanding neuroblastoma resistance to differentiation therapy and pave the way for hASH1-targeted therapies for augmenting the response of neuroblastoma to differentiation therapy. PMID:28066180

Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity. PMID:27738644

Most complex disease-associated genetic variants are located in non-coding regions and are therefore thought to be regulatory in nature. Association mapping of differential allelic expression (AE) is a powerful method to identify SNPs with direct cis-regulatory impact (cis-rSNPs). We used AE mapping to identify cis-rSNPs regulating gene expression in 55 and 63 HapMap lymphoblastoid cell lines from a Caucasian and an African population, respectively, 70 fibroblast cell lines, and 188 purified monocyte samples and found 40–60% of these cis-rSNPs to be shared across cell types. We uncover a new class of cis-rSNPs, which disrupt footprint-derived de novo motifs that are predominantly bound by repressive factors and are implicated in disease susceptibility through overlaps with GWAS SNPs. Finally, we provide the proof-of-principle for a new approach for genome-wide functional validation of transcription factor–SNP interactions. By perturbing NFκB action in lymphoblasts, we identified 489 cis-regulated transcripts with altered AE after NFκB perturbation. Altogether, we perform a comprehensive analysis of cis-variation in four cell populations and provide new tools for the identification of functional variants associated to complex diseases. PMID:25326100

Background Anchorage independent growth is an important hallmark of oncogenic transformation. Previous studies have shown that when adhesion dependent fibroblasts were prevented from adhering to a substrate they underwent anoikis. In the present study we have demonstrated how anoikis resistant cells gain the transformation related properties with sequential selection of genes. We have proposed this process as a model system for selection of transformed cells from normal cells. Results This report demonstrates that some fibroblasts can survive during late stages of anoikis, at which time they exhibit transformation-associated properties such as in vitro colony formation in soft agar and in vivo subcutaneous tumour formation in nude mice. Cytogenetic characterisation of these cells revealed that they contained a t (2; 2) derivative chromosome and they have a selective survival advantage in non adherent conditions. Gene expression profile indicated that these cells over expressed genes related to hypoxia, glycolysis and tumor suppression/metastasis which could be helpful in their retaining a transformed phenotype. Conclusion Our results reveal some new links between anoikis and cell transformation and they provide a reproducible model system which can potentially be useful to study multistage cancer and to identify new targets for drug development. PMID:21122158

Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in disease mechanisms and regenerative medicine. Epigenetic modifications enable differentiated cells to perpetuate molecular memory to retain their identity. Therefore, the aim of this study was to investigate the reprogramming modification of yak fibroblast cells that were permeabilized and incubated in the extracts of mesenchymal stem cells derived from mice adipose tissue [adipose-derived stem cells (ADSCs)]. According to the results, the treatment of ADSC extracts promoted colony formation. Moreover, pluripotent gene expression was associated with the loss of repressive histone modifications and increased global demethylation. The genes Col1a1 and Col1a2, which are typically found in differentiated cells only, demonstrated decreased expression and increased methylation in the 5'-flanking regulatory regions. Moreover, yak fibroblast cells that were exposed to ADSC extracts resulted in significantly different eight-cell and blastocyst formation rates of cloned embryos compared with their untreated counterparts. This investigation provides the first evidence that nuclear reprogramming of yak fibroblast cells is modified after the ADSC extract treatment. This research also presents a methodology for studying the dedifferentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells toward a pluripotent state without genetic alteration.

Early-passage rat kidney cells were immortalized or rescued from senescence with three different oncogenes: viral promoter-driven c-myc, H-ras (Val-12), and adenovirus type 5 E1a. The normal c-myc and H-ras (Gly-12) were unable to immortalize cells under similar conditions. Quantitation of RNA in the ras-immortalized lines demonstrated that the H-ras oncogene was expressed at a level equivalent to that of the normal H-ras gene in established human or rat cell lines. Cell lines immortalized by different oncogenes were found to have distinct growth responses to individual growth factors in a short-term assay. E1a-immortalized cells were largely independent of serum growth factors, whereas c-myc-immortalized cells responded to serum better than to epidermal growth factor and insulin. H-ras-immortalized cells responded significantly to insulin alone and gave a maximal response to epidermal growth factor and insulin. Several cellular genes associated with platelet-derived growth factor stimulation, including c-myc, were expressed at high levels in the H-ras-immortalized cells, and c-myc expression was deregulated, suggesting that the H-ras oncogene has provided a ''competence'' function. H-ras-immortalized cells could not be morphologically transformed by secondary transfection with a long terminal repeat-c-myc oncogene, but secondary transfection of the same cells with H-ras (Val-12) produced morphologically transformed colonies that had 20- to 40-fold higher levels of H-ras oncogene expression. Thus transformation in this system is dependent on high levels of H-ras oncogene expression rather than on the presence of activated H-ras and c-myc oncogenes in the same cell.

Utero-placental (Ut-Pl) angiogenesis and blood flow are fundamental for successful outcome of pregnancy. They are controlled by numerous vasodilator and vasoconstrictor systems such as endothelins (EDNs) and the renin angiotensin system. Dogs possess an invasive type of placentation, classified as endotheliochorial. Despite increasing knowledge regarding canine Ut-Pl function, little information exists on uterine and placental vascular activity during initiation, maintenance and termination of pregnancy in this species. The current study investigated expression of EDNs and their receptors (EDNRA and EDNRB) in the pre-implantation uterus and Ut-Pl compartments during gestation and at normal parturition, as well as in mid-pregnant dogs treated with the antigestagen aglepristone. The Ut-Pl mRNA expression of EDN1 and EDNRA was constant until mid-gestation and increased significantly during prepartum luteolysis. In contrast, EDN2 was highest pre-implantation and decreased following placentation, remaining low thereafter. Expression of the EDN-activating enzyme ECE1 and mRNA of EDNRB increased towards mid-gestation and was further elevated at prepartum luteolysis. Antigestagen treatment resulted in increased levels of EDN1 and EDNRA. At the cellular level, the uterine expression of EDN1, ECE1 and EDNRB was found predominantly in the endometrial surface and glandular epithelial cells; uterine signals for EDNRA were weak. In Ut-Pl all targets were mainly localized in the placenta fetalis, with syncytiotrophoblast staining stronger for ECE1 and EDNRB. In contrast, EDNRA stained strongly at the base of the placental labyrinth. Expression and localization of EDNs (EDN-1, -2), EDN receptors and ECE1 in the placenta fetalis suggests their involvement in the trophoblast invasion and proliferation.

The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B+ T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B+ B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role. PMID:24058482

The comparative study of cell types is a powerful approach toward deciphering animal evolution. To avoid selection biases, however, comparisons ideally involve all cell types present in a multicellular organism. Here, we use image registration and a newly developed “Profiling by Signal Probability Mapping” algorithm to generate a cellular resolution 3D expression atlas for an entire animal. We investigate three-segmented young worms of the marine annelid Platynereis dumerilii, with a rich diversity of differentiated cells present in relatively low number. Starting from whole-mount expression images for close to 100 neural specification and differentiation genes, our atlas identifies and molecularly characterizes 605 bilateral pairs of neurons at specific locations in the ventral nerve cord. Among these pairs, we identify sets of neurons expressing similar combinations of transcription factors, located at spatially coherent anterior-posterior, dorsal-ventral, and medial-lateral coordinates that we interpret as cell types. Comparison with motor and interneuron types in the vertebrate neural tube indicates conserved combinations, for example, of cell types cospecified by Gata1/2/3 and Tal transcription factors. These include V2b interneurons and the central spinal fluid-contacting Kolmer-Agduhr cells in the vertebrates, and several neuron types in the intermediate ventral ganglionic mass in the annelid. We propose that Kolmer-Agduhr cell-like mechanosensory neurons formed part of the mucociliary sole in protostome-deuterostome ancestors and diversified independently into several neuron types in annelid and vertebrate descendants. PMID:28584082

Heroin (3,6-diacetylmorphine) has various effects on the central nervous system with several neuropathological alterations including hypoxic-ischemic brain damage from respiratory depressing effects and neuroinflammatory response. Both of these mechanisms induce the release of cytokines, chemokines and other inflammatory mediators by the activation of many cell types such as leucocytes and endothelial and glial cells, especially microglia, the predominant immunocompetent cell type within the central nervous system. The aim of this study is to clarify the correlation between intravenous heroin administration in heroin related death and the neuroinflammatory response. We selected 45 cases among autopsies executed for heroin-related death (358 total cases); immunohistochemical studies and Western blotting analyses were used to investigate the expression of brain markers such as tumor necrosis factor-α, oxygen-regulated protein 150, (interleukins) IL-1β, IL-6, IL-8, IL-10, IL-15, cyclooxygenase-2, heat shock protein 70, and CD68 (MAC387). Findings demonstrated that morphine induces inflammatory response and cytokine release. In particular, oxygen-regulated protein 150, cyclooxygenase-2, heat shock protein 70, IL-6 and IL-15 cytokines were over-expressed with different patterns of cellularexpression. PMID:24084728

The comparative study of cell types is a powerful approach toward deciphering animal evolution. To avoid selection biases, however, comparisons ideally involve all cell types present in a multicellular organism. Here, we use image registration and a newly developed "Profiling by Signal Probability Mapping" algorithm to generate a cellular resolution 3D expression atlas for an entire animal. We investigate three-segmented young worms of the marine annelid Platynereis dumerilii, with a rich diversity of differentiated cells present in relatively low number. Starting from whole-mount expression images for close to 100 neural specification and differentiation genes, our atlas identifies and molecularly characterizes 605 bilateral pairs of neurons at specific locations in the ventral nerve cord. Among these pairs, we identify sets of neurons expressing similar combinations of transcription factors, located at spatially coherent anterior-posterior, dorsal-ventral, and medial-lateral coordinates that we interpret as cell types. Comparison with motor and interneuron types in the vertebrate neural tube indicates conserved combinations, for example, of cell types cospecified by Gata1/2/3 and Tal transcription factors. These include V2b interneurons and the central spinal fluid-contacting Kolmer-Agduhr cells in the vertebrates, and several neuron types in the intermediate ventral ganglionic mass in the annelid. We propose that Kolmer-Agduhr cell-like mechanosensory neurons formed part of the mucociliary sole in protostome-deuterostome ancestors and diversified independently into several neuron types in annelid and vertebrate descendants.

The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B(+) T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B(+) B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role.

Primary cutaneous mucinous carcinoma (PCMC) is a rare sweat gland tumor characterized by the presence of abundant mucin around the tumor islands, but the molecular mechanisms for this structure are not well elucidated. Because mucin is epithelial in nature, it is likely to be produced by epithelial tumor cells, not by surrounding stromal cells. We hypothesized that the abundant mucin is a result of reversed cellular polarity of the tumor. To test this hypothesis, we conducted an immunohistological study to investigate expression of tight junction (TJ) proteins occludin and ZO-1 in PCMC, as well as in normal sweat glands and other sweat gland tumors. Dot-like or linear expression of TJ proteins was observed at ductal structures of sweat glands, and ductal or cystic structures of related tumors. In PCMC, however, TJ protein expression was clearly visible at the edges of tumor cell islands. This study provides evidence to show that the characteristic histological structure of PCMC is caused by inverse polarization of the tumor cells, and that TJ proteins are useful markers of ductal differentiation in sweat gland tumors.

MUC1 mucin is aberrantly expressed in many epithelial malignancies and is a promising tumor antigen for target-directed immunotherapy against human breast cancer. Mycobacterium BCG is an effective immunoadjuvant which is known to induce Th1 immune response. Recombinant BCG expressing tumor antigen and secreting cytokine may therefore potentiate the tumor antigen-specific immune responses. In this study, we constructed a recombinant BCG-MUC1-IL2, which expresses a high level of human MUC1 VNTR core protein and secretes functional interleukin 2 (IL2). The immune responses induced by BCG-MUC1-IL2 were examined using a SCID mouse model reconstituted with immunologically competent human lymphocytes, SCID/hu-PBL. The mucin-specific IFN-gamma was secreted only by the lymphocytes derived from animals immunized with BCG-MUC1-IL2, but not with BCG-vector or purified mucin protein for the vaccination. In contrast, in vitro secretion of IL4 by the immunized lymphocytes was only seen in the group of animals which received native MUC1 protein, but not BCG-MUC1-IL2 and BCG-vector. Minimal MUC1-specific IgG and IgM were detected in SCID/hu-PBL mice vaccinated with BCG-MUC1-IL2. These results suggest that BCG-MUC1-IL2 preferentially induces MUC1-specific cellular immune responses and it may serve as a vaccine for breast cancer prevention and treatment.

Both folic acid (FA)- and methoxypoly(ethylene glycol) (mPEG)-conjugated chitosan nanoparticles (NPs) had been designed for targeted and prolong anticancer drug delivery system. The chitosan NPs were prepared with combination of ionic gelation and chemical cross-linking method, followed by conjugation with both FA and mPEG, respectively. FA-mPEG-NPs were compared with either NPs or mPEG-/FA-NPs in terms of their size, targeting cellular efficiency and tumor tissue distribution. The specificity of the mPEG-FA-NPs targeting cancerous cells was demonstrated by comparative intracellular uptake of NPs and mPEG-/FA-NPs by human adenocarcinoma HeLa cells. Mitomycin C (MMC), as a model drug, was loaded to the mPEG-FA-NPs. Results show that the chitosan NPs presented a narrow-size distribution with an average diameter about 200 nm regardless of the type of functional group. In addition, MMC was easily loaded to the mPEG-FA-NPs with drug-loading content of 9.1%, and the drug releases were biphasic with an initial burst release, followed by a subsequent slower release. Laser confocal scanning imaging proved that both mPEG-FA-NPs and FA-NPs could greatly enhance uptake by HeLa cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that an increased amount of mPEG-FA-NPs or FA-NPs were accumulated in the tumor tissue relative to the mPEG-NPs or NPs alone. These results suggest that both FA- and mPEG-conjugated chitosan NPs are potentially prolonged drug delivery system for tumor cell-selective targeting treatments.

Both folic acid (FA)- and methoxypoly(ethylene glycol) (mPEG)-conjugated chitosan nanoparticles (NPs) had been designed for targeted and prolong anticancer drug delivery system. The chitosan NPs were prepared with combination of ionic gelation and chemical cross-linking method, followed by conjugation with both FA and mPEG, respectively. FA-mPEG-NPs were compared with either NPs or mPEG-/FA-NPs in terms of their size, targeting cellular efficiency and tumor tissue distribution. The specificity of the mPEG-FA-NPs targeting cancerous cells was demonstrated by comparative intracellular uptake of NPs and mPEG-/FA-NPs by human adenocarcinoma HeLa cells. Mitomycin C (MMC), as a model drug, was loaded to the mPEG-FA-NPs. Results show that the chitosan NPs presented a narrow-size distribution with an average diameter about 200 nm regardless of the type of functional group. In addition, MMC was easily loaded to the mPEG-FA-NPs with drug-loading content of 9.1%, and the drug releases were biphasic with an initial burst release, followed by a subsequent slower release. Laser confocal scanning imaging proved that both mPEG-FA-NPs and FA-NPs could greatly enhance uptake by HeLa cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that an increased amount of mPEG-FA-NPs or FA-NPs were accumulated in the tumor tissue relative to the mPEG-NPs or NPs alone. These results suggest that both FA- and mPEG-conjugated chitosan NPs are potentially prolonged drug delivery system for tumor cell-selective targeting treatments. PMID:22027239

Both folic acid (FA)- and methoxypoly(ethylene glycol) (mPEG)-conjugated chitosan nanoparticles (NPs) had been designed for targeted and prolong anticancer drug delivery system. The chitosan NPs were prepared with combination of ionic gelation and chemical cross-linking method, followed by conjugation with both FA and mPEG, respectively. FA-mPEG-NPs were compared with either NPs or mPEG-/FA-NPs in terms of their size, targeting cellular efficiency and tumor tissue distribution. The specificity of the mPEG-FA-NPs targeting cancerous cells was demonstrated by comparative intracellular uptake of NPs and mPEG-/FA-NPs by human adenocarcinoma HeLa cells. Mitomycin C (MMC), as a model drug, was loaded to the mPEG-FA-NPs. Results show that the chitosan NPs presented a narrow-size distribution with an average diameter about 200 nm regardless of the type of functional group. In addition, MMC was easily loaded to the mPEG-FA-NPs with drug-loading content of 9.1%, and the drug releases were biphasic with an initial burst release, followed by a subsequent slower release. Laser confocal scanning imaging proved that both mPEG-FA-NPs and FA-NPs could greatly enhance uptake by HeLa cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that an increased amount of mPEG-FA-NPs or FA-NPs were accumulated in the tumor tissue relative to the mPEG-NPs or NPs alone. These results suggest that both FA- and mPEG-conjugated chitosan NPs are potentially prolonged drug delivery system for tumor cell-selective targeting treatments.

Because the olfactory system plays a major role in food consumption, and because “food addiction” and associated morbidities have reached epidemic proportions, we tested the hypothesis that dietary energy restriction can modify adverse effects of cocaine on behavior and olfactory cellular and molecular plasticity. Mice maintained on an alternate day fasting (ADF) diet exhibited increased baseline locomotion and increased cocaine-sensitized locomotion during cocaine conditioning, despite no change in cocaine conditioned place preference, compared to mice fed ad libitum. Levels of dopamine and its metabolites in the olfactory bulb (OB) were suppressed in mice on the ADF diet compared to mice on the control diet, independent of acute or chronic cocaine treatment. The expression of several enzymes involved in dopamine metabolism including tyrosine hydroxylase, monoamine oxidases A and B (MAOA), and catechol-O-methyltransferase were significantly reduced in OBs of mice on the ADF diet. Both acute and chronic administration of cocaine suppressed the production of new OB cells, and this effect of cocaine was attenuated in mice on the ADF diet. Cocaine administration to mice on the control diet resulted in up-regulation of OB genes involved in mitochondrial energy metabolism, synaptic plasticity, cellular stress responses, and calcium- and cyclic AMP-mediated signaling, whereas multiple olfactory receptor genes were down-regulated by cocaine treatment. ADF abolished many of the effects of cocaine on OB gene expression. Our findings reveal that dietary energy intake modifies the neural substrates underlying some of the behavioral and physiological responses to repeated cocaine treatment, and also suggest novel roles for the olfactory system in addiction. The data further suggest that modification of dietary energy intake could provide a novel potential approach to addiction treatments. PMID:20456017

Reading frame maintenance is a critical property of ribosomes. However, a number of genetic elements have been described that can induce ribosomes to shift on mRNAs, the most well understood of which are a class that directs ribosomal slippage by one base in 5′ (-1) direction. This is referred to as programmed -1 ribosomal frameshifting (-1 PRF). Recently, a new -1 PRF promoting element was serendipitously discovered in a study examining the effects of stretches of adenosines in the coding sequences of mRNAs. Here, we discuss this finding, recent studies describing how -1 PRF is used to control gene expression in eukaryotes, and how -1 PRF is itself regulated. The implications of dysregulation of -1 PRF on human health are examined, as are possible new areas in which novel -1 PRF promoting elements might be discovered. PMID:26661048

Cell gene expression is affected by both the kind and mode of growth factor stimulation (diffusive vs. non-diffusive). Epidermal growth factor (EGF) was pattern-immobilized on a polystyrene plate. Although the growth of the rat phaeochromocytoma cell line PC12 is stimulated by diffusible EGF, and differentiation is stimulated by diffusible nerve growth factor (NGF), immobilized (non-diffusible) EGF stimulated PC12 differentiation. The immobilized EGF caused a long-lasting stimulation of the intracellular signal protein mitogen-associated protein MAP kinase (MAPK, also known as ERK) and p38 (a subfamily of the MAPK superfamily) in cells, as did diffusible NGF. The switching between growth stimulation and differentiation is considered to be due to the duration of the stimulus. The function of the biosignal conjugate was regulated using conjugation methodology.

The effect of the uncoupler of oxidative phosphorylation, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), on the tight junction of Madin-Darby canine kidney cells was examined. FCCP induced an abrupt decrease in the transepithelial electrical resistance of the confluent monolayers over a period of 20 s. When FCCP was withdrawn from the incubation medium, the monolayer resistance recovered to close to the original level in less than 2 h. Staining of the tight junction-associated protein ZO-1 showed that the changes in transepithelial electrical resistance were accompanied by a diffusing of the protein away from cell peripheries and a reconcentration to the tight junction areas following resistance recovery. Intracellular pH was decreased by FCCP on a similar time-scale with no obvious changes in ATP levels over this time-course. These data suggest that the uncoupler FCCP has a profound effect on tight junction permeability and cellulardistribution of the tight junction protein ZO-1 in the epithelial cells and that it probably acts by breaking down proton gradients and altering intracellular pH.

ABSTRACT Neural tube closure is a critical developmental event that relies on actomyosin contractility to facilitate specific processes such as apical constriction, tissue bending, and directional cell rearrangements. These complicated processes require the coordinated activities of Rho-Kinase (Rock), to regulate cytoskeletal dynamics and actomyosin contractility, and the Planar Cell Polarity (PCP) pathway, to direct the polarized cellular behaviors that drive convergent extension (CE) movements. Here we investigate the role of Shroom3 as a direct linker between PCP and actomyosin contractility during mouse neural tube morphogenesis. In embryos, simultaneous depletion of Shroom3 and the PCP components Vangl2 or Wnt5a results in an increased liability to NTDs and CE failure. We further show that these pathways intersect at Dishevelled, as Shroom3 and Dishevelled 2 co-distribute and form a physical complex in cells. We observed that multiple components of the Shroom3 pathway are planar polarized along mediolateral cell junctions in the neural plate of E8.5 embryos in a Shroom3 and PCP-dependent manner. Finally, we demonstrate that Shroom3 mutant embryos exhibit defects in planar cell arrangement during neural tube closure, suggesting a role for Shroom3 activity in CE. These findings support a model in which the Shroom3 and PCP pathways interact to control CE and polarized bending of the neural plate and provide a clear illustration of the complex genetic basis of NTDs. PMID:25596276

Addition of nicotine causes a dose- and time-dependent inhibition of cell growth in established human and murine cells. In the human promyelocytic HL-60 leukemic cells, 3 mM nicotine results in a 50% inhibition of cellular proliferation after 80 h. Nicotine was also found to affect the cell cycle distribution of HL-60 cells. Treatment with 4 mM nicotine for 20 h causes an increase in proportion of Gl-phase cells (from 49% to 57%) and a significant decrease in the proportion of S-phase cells (from 41% to 32%). These results suggest that nicotine causes cell arrest in the Gl-phase which may in part account for its effects on cell growth. To determine whether nicotine has a primary effect on the uptake/transport of macromolecular precursors into cells, HL-60 cells were treated with 2-6 mM nicotine for 30 h/sub 3/ at the end of which time cells were labeled with (/sup 3/H)thymidine, (/sup 3/H)uridine, (/sup 14/C)lysine and (/sup 35/S)methionine, the trichloroacetic acid (TCA) soluble and insoluble radioactivities from each of the labeling conditions were determined. These studies show that nicotine primarily affect the synthesis of proteins.

The distribution and size of a surface membrane antigen identified by a monoclonal antibody (MAC9393) have been examined in testicular and epididymal bovine sperm preparations. Western blots indicated a substantial decrease in molecular mass of the antigen during epididymal maturation from approximately 87 kDa in the testis to approximately 35 kDa in the cauda epididymidis. This was accompanied by a change in its cellular localization from the neck and whole head to the acrosomal region. N-terminal microsequencing identified MAC393 antigen as the beta-chain of clusterin. A polyclonal antiserum to the alpha-chain of clusterin recognized both testicular and epididymal forms and revealed that the heterodimer was present on the sperm tail as well as the acrosome. These findings are explained by the co-existence of dimeric and monomeric pools of clusterin on spermatozoa. The polyclonal antiserum recognizes both testicular and epididymal forms of the heterodimer and although the monoclonal antibody binds to the testicular heterodimer, it only recognizes the beta-chain monomer of epididymal clusterin. These findings support previous observations made on human spermatozoa that two forms of clusterin, the beta-chain monomer and the heterodimer, are present on the surface membrane and in seminal plasma.

Segment 5 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP5, was tagged with glutathione S-transferase and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not internalized, which indicates it is not involved in receptor-mediated endocytosis. The purified VP5 protein was shown to be able to permeabilize mammalian and Culicoides insect cells, inducing cytotoxicity. Sequence analysis revealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. To assess the role of each feature in the observed cytotoxicity, a series of deleted VP5 molecules were generated, and their expression and biological activity was compared with the parental molecule. VP5 derivatives that included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane destabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing the adjacent downstream sequences were also assessed for their effect on the cell membrane. Both helices, but not the downstream VP5 sequence, exhibited cytotoxicity with the most-amino-terminal helix (aa 1 to 20) showing a higher activity than the adjacent peptide (aa 22 to 41). Purified VP5 was shown to readily form trimers in solution, a feature of many proteins involved in membrane penetration. Taken together, these data support a role for VP5 in virus-cell penetration consistent with its revelation in the entry vesicle subsequent to cell binding and endocytosis. PMID:11507181

Polyamines are important in cell growth and wound repair, but have also been implicated in inflammation-induced carcinogenesis. Polyamine metabolism includes back-conversion of spermine to spermidine by the enzyme spermine oxidase (SMO), which produces hydrogen peroxide that causes oxidative stress. In ulcerative colitis (UC), levels of spermine are decreased compared to spermidine. Therefore, we sought to determine if SMO is involved in UC. Colon biopsies and clinical information from subjects undergoing colonoscopy for evaluation of UC or colorectal cancer screening were utilized from 16 normal controls and 53 UC cases. Histopathologic disease severity was graded and the Mayo Disease Activity Index (DAI) and endoscopy subscore assessed. SMO mRNA expression was measured in frozen biopsies by TaqMan-based real-time polymerase chain reaction (PCR). Formalin-fixed tissues were used for SMO immunohistochemistry. There was a 3.1-fold upregulation of SMO mRNA levels in UC patients compared to controls (P = 0.044), and a 3.7-fold increase in involved left colon versus paired uninvolved right colon (P < 0.001). With worsening histologic injury in UC there was a progressive increase in SMO staining of mononuclear inflammatory cells. There was a similar increase in SMO staining with worsening endoscopic disease severity and strong correlation with the DAI (r = 0.653, P < 0.001). Inflammatory cell SMO staining was increased in involved left colon versus uninvolved right colon. SMO expression is upregulated in UC tissues, deriving from increased levels in mononuclear inflammatory cells. Dysregulated polyamine homeostasis may contribute to chronic UC by altering immune responses and increasing oxidative stress.

Background Polyamines are important in cell growth and wound repair, but have also been implicated in inflammation-induced carcinogenesis. Polyamine metabolism includes back-conversion of spermine to spermidine by the enzyme spermine oxidase (SMO), which produces hydrogen peroxide that causes oxidative stress. In ulcerative colitis (UC), levels of spermine are decreased compared to spermidine. Therefore, we sought to determine if SMO is involved in UC. Methods Colon biopsies and clinical information from subjects undergoing colonoscopy for evaluation of UC or colorectal cancer screening were utilized from 16 normal controls and 53 UC cases. Histopathologic disease severity was graded and the Mayo Disease Activity Index (DAI) and endoscopy subscore assessed. SMO mRNA expression was measured in frozen biopsies by Taq-Man-based real-time polymerase chain reaction (PCR). Formalin-fixed tissues were used for SMO immunohistochemistry. Results There was a 3.1-fold upregulation of SMO mRNA levels in UC patients compared to controls (P = 0.044), and a 3.7-fold increase in involved left colon versus paired uninvolved right colon (P < 0.001). With worsening histologic injury in UC there was a progressive increase in SMO staining of mononuclear inflammatory cells. There was a similar increase in SMO staining with worsening endoscopic disease severity and strong correlation with the DAI (r = 0.653, P < 0.001). Inflammatory cell SMO staining was increased in involved left colon versus uninvolved right colon. Conclusions SMO expression is upregulated in UC tissues, deriving from increased levels in mononuclear inflammatory cells. Dysregulated polyamine homeostasis may contribute to chronic UC by altering immune responses and increasing oxidative stress. PMID:20127992

In humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene we investigated the effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol, mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the level of glycerophospholipids. PMID:20041128

The distribution of possible generation times in mammalian cells does not appear to be continous within the limits of range for each cell type; rather, generation time is quantized in multiples of 3-4 hr. Synchronous cultures of Chinese hamster V79 cells were prepared using manual and automated methods to select and stage mitotic cells. Using synchronous cultures and time-lapse video tape microscopy, it was possible to show that generation times within a population of mitotically selected cells normally disperse in a quantized fashion, with intervals of 3-4 hr occurring between bursts in division. In addition, at temperatures above 37 degrees, V79 cells have a 7.5-8.5 hr modal cell cycle, while at temperatures from 36.5 degrees to 33.5 degrees the modal cell cycle is 11-12 hr long. A survey of the synchrony literature reveals that the tendency to preferred generation times holds between cell lines. The distribution of modal generation times from a variety of different cell types forms a series with a similar interval but with a greater range of values than that observed here for V79 cells. To satisfy the published data and the work presented here, I propose a subcycle, Gq, which has a traverse time equal to the period of the clock. The period appears to be fixed at close to the same value in all mammalian somatic cells. The timekeeping mechanism appears to be temperature compensated, since the time required to traverse Gq is constant at temperatures between 34 degrees and 39 degrees. It is suggested that cell cycle time increases at lower temperatures, lower serum concentration, and high cell densitite because the number of rounds of traverse through Gq increases. PMID:1069287

Prediction of possible flux distributions in a metabolic network provides detailed phenotypic information that links metabolism to cellular physiology. To estimate metabolic steady-state fluxes, the most common approach is to solve a set of macroscopic mass balance equations subjected to stoichiometric constraints while attempting to optimize an assumed optimal objective function. This assumption is justifiable in specific cases but may be invalid when tested across different conditions, cell populations, or other organisms. With an aim to providing a more consistent and reliable prediction of flux distributions over a wide range of conditions, in this article we propose a framework that uses the flux minimization principle to predict active metabolic pathways from mRNA expression data. The proposed algorithm minimizes a weighted sum of flux magnitudes, while biomass production can be bounded to fit an ample range from very low to very high values according to the analyzed context. We have formulated the flux weights as a function of the corresponding enzyme reaction's gene expression value, enabling the creation of context-specific fluxes based on a generic metabolic network. In case studies of wild-type Saccharomyces cerevisiae, and wild-type and mutant Escherichia coli strains, our method achieved high prediction accuracy, as gauged by correlation coefficients and sums of squared error, with respect to the experimentally measured values. In contrast to other approaches, our method was able to provide quantitative predictions for both model organisms under a variety of conditions. Our approach requires no prior knowledge or assumption of a context-specific metabolic functionality and does not require trial-and-error parameter adjustments. Thus, our framework is of general applicability for modeling the transcription-dependent metabolism of bacteria and yeasts.

Hepcidin is a peptide hormone belonging to the defensin family of cationic antimicrobial molecules that has an essential role in systemic iron homeostasis. The peptide is synthesised by hepatocytes and transported in the circulation to target tissues where it regulates the iron export function of the ferrous iron permease, ferroportin. In the brain hepcidin protein has been identified using immuno-histochemistry and mRNA by real-time PCR but not by in situ hybridisation raising the question of whether there is measurable transcription of the hepcidin gene in the central nervous system. Alternatively hepcidin could be transported as a hormone to the brain via the circulation. By RT-PCR hepcidin mRNA was present at low level throughout normal rat brain while in situ hybridisation to detect low-abundant mRNA revealed that transcripts were restricted to endothelium of blood vessels and choroid plexus. In contrast, hepcidin protein analysed by immuno-histochemistry was highly expressed in blood vessels, in endothelium and in pericytes. Hepcidin was also present in glial cells and in the olfactory bulb, sub-ventricular zone and dentate gyrus, areas where neurogenesis and synaptic plasticity are maintained throughout adult life. The hepcidin species identified by Western blotting in sub-ventricular zone, cortex and hippocampus migrated as a ~2.8 kDa band, identical in size to hepcidin present in normal rat serum suggesting that hepcidin in brain was the full-length biologically active 25 amino acid peptide. Hepcidin co-localised with ferroportin in ependymal cells of the sub-ventricular zone and in the corpus callosum consistent with a regulatory role in iron metabolism at these sites. Hepcidin protein was widely expressed in brain parenchyma while levels of hepcidin gene transcription appeared to be below the limits of detection of the in situ hybridisation probes. This disparity suggests that not all hepcidin in the brain is transcribed in situ and may originate in part

We present a human miRNA tissue atlas by determining the abundance of 1997 miRNAs in 61 tissue biopsies of different organs from two individuals collected post-mortem. One thousand three hundred sixty-four miRNAs were discovered in at least one tissue, 143 were present in each tissue. To define the distribution of miRNAs, we utilized a tissue specificity index (TSI). The majority of miRNAs (82.9%) fell in a middle TSI range i.e. were neither specific for single tissues (TSI > 0.85) nor housekeeping miRNAs (TSI < 0.5). Nonetheless, we observed many different miRNAs and miRNA families that were predominantly expressed in certain tissues. Clustering of miRNA abundances revealed that tissues like several areas of the brain clustered together. Considering -3p and -5p mature forms we observed miR-150 with different tissue specificity. Analysis of additional lung and prostate biopsies indicated that inter-organism variability was significantly lower than inter-organ variability. Tissue-specific differences between the miRNA patterns appeared not to be significantly altered by storage as shown for heart and lung tissue. MiRNAs TSI values of human tissues were significantly (P = 10−8) correlated with those of rats; miRNAs that were highly abundant in certain human tissues were likewise abundant in according rat tissues. We implemented a web-based repository enabling scientists to access and browse the data (https://ccb-web.cs.uni-saarland.de/tissueatlas). PMID:26921406

We present a human miRNA tissue atlas by determining the abundance of 1997 miRNAs in 61 tissue biopsies of different organs from two individuals collected post-mortem. One thousand three hundred sixty-four miRNAs were discovered in at least one tissue, 143 were present in each tissue. To define the distribution of miRNAs, we utilized a tissue specificity index (TSI). The majority of miRNAs (82.9%) fell in a middle TSI range i.e. were neither specific for single tissues (TSI > 0.85) nor housekeeping miRNAs (TSI < 0.5). Nonetheless, we observed many different miRNAs and miRNA families that were predominantly expressed in certain tissues. Clustering of miRNA abundances revealed that tissues like several areas of the brain clustered together. Considering -3p and -5p mature forms we observed miR-150 with different tissue specificity. Analysis of additional lung and prostate biopsies indicated that inter-organism variability was significantly lower than inter-organ variability. Tissue-specific differences between the miRNA patterns appeared not to be significantly altered by storage as shown for heart and lung tissue. MiRNAs TSI values of human tissues were significantly (P = 10(-8)) correlated with those of rats; miRNAs that were highly abundant in certain human tissues were likewise abundant in according rat tissues. We implemented a web-based repository enabling scientists to access and browse the data (https://ccb-web.cs.uni-saarland.de/tissueatlas).

Background and Objectives: Major abdominal procedures are strongly associated with postoperative immunosuppression and subsequent increased patient morbidity. It is believed that laparoscopic surgery causes less depletion of the systemic immune function because of the reduced tissue trauma. Various cytokines and monocytic HLA-DR expression have been successfully implemented to assess postoperative immune function. The aim of our study was to show the difference in immunologic profiles after minimally invasive versus conventional liver resection. Methods: Ten animals underwent either laparoscopic or conventional open left lateral liver resection. Flow cytometric characteristics of HLA-DR expression on monocytes and lipopolysaccharide-stimulated cellular secretion of tumor necrosis factor α, interferon γ, interleukin 6, and interleukin 8 were measured and analyzed in ex vivo whole blood samples. Intraoperative and postoperative clinical outcome parameters were also documented and evaluated. Results: All animals survived the procedures. Postoperative complications were fever (n = 3), wound infections (n = 2), and biloma (n = 1). Open surgery showed a morbidity rate of 80% compared with 40% after laparoscopic surgery. Laparoscopic liver resection showed no postoperative immunoparalysis. Major histocompatibility complex class II expression in this group was elevated, whereas the open surgery group showed decreased major histocompatibility complex class II expression on postoperative day 1. Postoperative secretion of tumor necrosis factor α, interleukin 6, and interferon γ was lower in the open surgery group. Elevated transaminase levels after laparoscopy might have resulted from an ischemia/reperfusion injury caused by the capnoperitoneum. Conclusion: Major immunoparalysis depression was not observed in either group. Laparoscopic surgery shows a tendency to improve immunologic recovery after liver resection. PMID:24398205

SUMMARY Aflatoxin biosynthesis in Aspergillus parasiticus requires at least 17 enzyme activities (from acetate). Although the activities of most aflatoxin biosynthetic enzymes have been established, the mechanisms that govern transport and sub-cellular localization of these enzymes are not clear. We developed plasmid constructs that express Nor-1 fused to a green fluorescent protein reporter (EGFP) to monitor transport and localization of this early pathway enzyme in real time in Aspergillus parasiticus. Plasmids expressing EGFP fused to Nor-1 were introduced into A. parasiticus B62 (carries non-functional Nor-1). Transformants were screened for increased aflatoxin accumulation (restored Nor-1 activity) on coconut agar medium and for EGFP expression using fluorescence microscopy. Increased aflatoxin accumulation was confirmed by TLC and ELISA. Nor-1 fused to EGFP at either the N- or C- terminus functionally complemented non-functional Nor-1 in B62 and increased aflatoxin synthesis to wild-type (N-terminus) or lower levels (C-terminus). We detected full-length Nor-1 fusion proteins in transformants with increased aflatoxin accumulation (Western blot) and determined that the expression plasmid integrated at the nor-1 locus in these cells (Southern blot). Confocal laser scanning microscopy (CLSM) demonstrated that Nor-1 fusion proteins localized in the cytoplasm and vacuoles of fungal hyphae grown on aflatoxin-inducing solid media for 48 h; control EGFP (no Nor-1) did not localize to vacuoles until 72 h. The highest rate of aflatoxin synthesis coincided with the highest rate of transport of Nor-1 fusion proteins to the vacuole strongly suggesting that Nor-1 is synthesized in the cytoplasm and transported to the vacuole to carry out an early step in aflatoxin synthesis. PMID:19217941

The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed “ghost” cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets. PMID:26954570

Vitamin C has been shown to delay the cellular senescence and was considered a candidate for chemoprevention and cancer therapy. To understand the reported contrasting roles of vitamin C: growth-promoting in the primary cells and growth-inhibiting in cancer cells, primary mouse embryonic fibroblasts (MEF) and their isogenic spontaneously immortalized fibroblasts with unlimited cell division potential were used as the model pair. We used microarray gene expression profiling to show that the immortalized MEF possess human cancer gene expression fingerprints including a pattern of up-regulation of inflammatory response-related genes. Using the MEF model, we found that a physiological treatment level of vitamin C (10(-5) M), but not other unrelated antioxidants, enhanced cell growth. The growth-promoting effect was associated with a pattern of enhanced expression of cell cycle- and cell division-related genes in both primary and immortalized cells. In the immortalized MEF, physiological treatment levels of vitamin C also enhanced the expression of immortalization-associated genes including a down-regulation of genes in the extracellular matrix functional category. In contrast, confocal immunofluorescence imaging of the primary MEF suggested an increase in collagen IV protein upon vitamin C treatment. Similar to the cancer cells, the growth-inhibitory effect of the redox-active form of vitamin C was preferentially observed in immortalized MEF. All effects of vitamin C required its intracellular presence since the transporter-deficient SVCT2-/- MEF did not respond to vitamin C. SVCT2-/- MEF divided and became immortalized readily indicating little dependence on vitamin C for the cell division. Immortalized SVCT2-/- MEF required higher concentration of vitamin C for the growth inhibition compared to the immortalized wildtype MEF suggesting an intracellular vitamin C toxicity. The relevance of our observation in aging and human cancer prevention was discussed.

Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

CD8+ T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA) utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8+ T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens. PMID:22761956

At presentation, most cases of adenocarcinoma of the esophagus (ACE) are inoperable. Although chemotherapy can prolong survival, patients eventually die as a result of refractory disease. Epidermal growth factor receptor (EGFR) is almost universally expressed in ACE and is a negative prognostic factor. This open-label, two-center, noncomparative, two-part phase II trial assessed the EGFR tyrosine kinase inhibitor gefitinib (500 mg/d) in patients with advanced, inoperable ACE. The primary end point was tumor response. The effect of EGFR inhibition was also evaluated by gene expression analysis of tumor biopsies taken before gefitinib treatment and 28 days after. Twenty-seven patients were recruited and evaluable for tumor response and safety. Three patients had a partial response and seven had stable disease, giving a disease control rate (partial response + stable disease) of 37%. Drug-related adverse events were generally mild: diarrhea in 19 (grade 3 in three) and rash in 19 (grade 3 in five) patients, and there were no grade 4 drug-related adverse events. Microarray experiments on tumor biopsies showed that gefitinib also down-regulated oncogenes associated with tumor progression. Ki67 (a marker of tumor growth) expression decreased in five of seven biopsies taken before and after treatment. Gefitinib (500 mg/d) is an active and generally well-tolerated treatment for ACE. Studies on endoscopic biopsies are feasible and indicate that gefitinib inhibits both gene expression and cellular biology at 500 mg/d, and these may provide surrogate end points for predictive biomarkers. Further trials of gefitinib are warranted, particularly as patient response seems to be durable and current second-line chemotherapy options have no proven ability to prolong life.

Cohesion between sister chromatids is essential for faithful chromosome segregation. In budding yeast, the acetyltransferase Eco1/Ctf7 establishes cohesion during DNA replication in S phase and in response to DNA double strand breaks in G2/M phase. In humans two Eco1 orthologs exist: ESCO1 and ESCO2. Both proteins are required for proper sister chromatid cohesion, but their exact function is unclear at present. Since ESCO2 has been identified as the gene defective in the rare autosomal recessive cohesinopathy Roberts syndrome (RBS), cells from RBS patients can be used to elucidate the role of ESCO2. We investigated for the first time RBS cells in comparison to isogenic controls that stably express V5- or GFP-tagged ESCO2. We show that the sister chromatid cohesion defect in the transfected cell lines is rescued and suggest that ESCO2 is regulated by proteasomal degradation in a cell cycle-dependent manner. In comparison to the corrected cells RBS cells were hypersensitive to the DNA-damaging agents mitomycin C, camptothecin and etoposide, while no particular sensitivity to UV, ionizing radiation, hydroxyurea or aphidicolin was found. The cohesion defect of RBS cells and their hypersensitivity to DNA-damaging agents were not corrected by a patient-derived ESCO2 acetyltransferase mutant (W539G), indicating that the acetyltransferase activity of ESCO2 is essential for its function. In contrast to a previous study on cells from patients with Cornelia de Lange syndrome, another cohesinopathy, RBS cells failed to exhibit excessive chromosome aberrations after irradiation in G2 phase of the cell cycle. Our results point at an S phase-specific role for ESCO2 in the maintenance of genome stability.

We have previously reported that 28-day treatment with hepatocarcinogens increases liver cells expressing p21(Cip1), a G1/S checkpoint protein, and M phase proteins, i.e., nuclear Cdc2, Aurora B, phosphorylated-Histone H3 (p-Histone H3) and heterochromatin protein 1α (HP1α), in rats. To examine the roles of these markers in the early stages of carcinogenesis, we investigated their cellulardistribution in several carcinogenic target organs using rat two-stage carcinogenesis models. Promoting agents targeting the liver (piperonyl butoxide and methapyrilene hydrochloride), thyroid (sulfadimethoxine), urinary bladder (phenylethyl isothiocyanate), and forestomach and glandular stomach (catechol) were administered to rats after initiation treatment for the liver with N-diethylnitrosamine, thyroid with N-bis(2-hydroxypropyl)nitrosamine, urinary bladder with N-butyl-N-(4-hydroxybutyl)nitrosamine, and forestomach and glandular stomach with N-methyl-N'-nitro-N-nitrosoguanidine. Numbers of cells positive for nuclear Cdc2, Aurora B, p-Histone H3 and HP1α increased within preneoplastic lesions as determined by glutathione S-transferase placental form in the liver or phosphorylated p44/42 mitogen-activated protein kinase in the thyroid, and hyperplastic lesions having no known preneoplastic markers in the urinary bladder, forestomach and glandular stomach. Immunoreactive cells for p21(Cip1) were decreased within thyroid preneoplastic lesions; however, they were increased within liver preneoplastic lesions and hyperplastic lesions in other organs. These results suggest that M phase disruption commonly occur during the formation of preneoplastic lesions and hyperplastic lesions. Differences in the expression patterns of p21(Cip1) between thyroid preneoplastic and proliferative lesions in other organs may reflect differences in cell cycle regulation involving G1/S checkpoint function between proliferative lesions in each organ.

Human T cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that can cause adult T-cell leukemia (ATL) and other diseases. The HTLV-1 bZIP factor (HBZ), which is encoded by an mRNA of the opposite polarity of the viral genomic RNA, interacts with several transcription factors and is involved in T cell proliferation, viral gene transcription and cellular transformation. Cyclin D1 is a pivotal regulatory protein involved in cell cycle progression, and its depressed expression correlates with cell cycle prolongation or arrested at the G1/S transition. In our present study, we observed that HBZ expression suppressed cyclin D1 level. To investigate the role of HBZ on cyclin D1 depression, we transduced HBZ with lentivirus vector into 293T cells, CEM cells and Jurkat cells. The results of Western blot, RT-PCR and luciferase assays showed that transcriptional activity of the cyclin D1 promoter was suppressed by the bZIP domain of HBZ (HBZ-bZIP) through cyclic AMP response element (CRE) site. Immunoprecipitation and GST pull-down assays showed the binding of HBZ-bZIP to CRE-binding protein (CREB), which confirmed that the cyclin D1 promoter activity inhibition via the CRE-site was mediated by HBZ-bZIP. The results suggested that HBZ suppressed cyclin D1 transcription through interactions with CREB and along with other viral protein, HBZ may play a causal role for leukemogenesis.

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis. PMID:24473084

Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis.

A large number of studies have focused on identifying molecular biomarkers, including microRNAs (miRNAs) to aid in the diagnosis and prognosis of the most common subtypes of non-Hodgkin lymphoma (NHL), Diffuse Large B-cell Lymphoma and Follicular Lymphoma. NHL is difficult to diagnose and treat with many cases becoming resistant to chemotherapy, hence the need to identify improved biomarkers to aid in both diagnosis and treatment modalities. This review summarises more recent research on the dysregulated miRNA expression profiles found in NHL, as well as the regulatory role and biomarker potential of cellular and circulating miRNAs found in tissue and serum, respectively. In addition, the emerging field of research focusing on miRNA single nucleotide polymorphisms (miRSNPs) in genes of the miRNA biogenesis pathway, in miRNA genes themselves, and in their target sites may provide new insights on gene expression changes in these genes. These miRSNPs may impact miRNA networks and have been shown to play a role in a host of different cancer types including haematological malignancies. With respect to NHL, a number of SNPs in miRNA-binding sites in target genes have been shown to be associated with overall survival. PMID:27999330

The newly synthesized calcium channel blocker, Ro44-5912, significantly potentiates doxorubicin (Dox)-induced cytotoxicity at non-cytotoxic concentrations in Dox-resistant human ovarian cell line, A2780/DX5, overexpressing P170-glycoprotein (Pgp). Induction of DNA single- and double-strand breaks (ssbs and dsbs) was measured using alkaline elution and constant-field gel electrophoresis (CFGE) assays. The results indicate that potentiation of the cytotoxicity of Dox by Ro44-5912 was accompanied by significant increases in both, Dox-induced DNA ssbs and dsbs in the resistant cells. Pulsed-field gel electrophoresis (PFGE) analysis showed that Dox induced DNA fragments in the 50-800 kilobase (kb) and 0.8-5.7 megabase (Mb) ranges. The majority of the newly synthesized DNA fragments were in the 50-800 kb range. Ro44-5912 treatment resulted in significant potentiation of DNA fragmentation in the 50-800 kb range with a minor increase in 0.8-5.7 Mb DNA fragments, suggesting that the modulator functions by potentiating nascent DNA fragmentation in the resistant cells. Exposure to Dox with Ro44-5912 was associated with a prolonged blockage of cells in the S-phase. In contrast, exposure to Dox alone resulted in temporary blockage of cells in G2/M phase (approximately 24 h) followed by restoration of cell proliferation and normal DNA histograms at 48 h after 2 h drug exposure. Incorporation of BrdUrd by flow cytometric analysis was inhibited by Dox in the presence of Ro44-5912, showing that there is a block of DNA replication. An increased damage in newly synthesized DNA could concur with a blocked DNA replication. Moreover, slowing progression through the S-phase in cells exposed to Dox in combination with Ro44-5912 is accompanied by increased sensitivity of Dox poisons, indicating a correlation of specific S-phase perturbation with the reversal of Dox resistance by Ro44-5912 in cells expressing Pgp. The results suggest that drug-induced augmentation of nascent DNA

The prefrontal cortex (PFC) is involved in behavioural control and cognitive processes that are altered in schizophrenia. The brainstem monoaminergic systems control PFC function, yet the cells/networks involved are not fully known. Serotonin (5-HT) and norepinephrine (NE) increase PFC neuronal activity through the activation of α(1)-adrenergic receptors (α(1)ARs) and 5-HT(2A) receptors (5-HT(2A)Rs), respectively. Neurochemical and behavioural interactions between these receptors have been reported. Further, classical and atypical antipsychotic drugs share nm in vitro affinity for α(1)ARs while having preferential affinity for D(2) and 5-HT(2A)Rs, respectively. Using double in situ hybridization we examined the cellularexpression of α(1)ARs in pyramidal (vGluT1-positive) and GABAergic (GAD(65/67)-positive) neurons in rat PFC and their co-localization with 5-HT(2A)Rs. α(1)ARs are expressed by a high proportion of pyramidal (59-85%) and GABAergic (52-79%) neurons. The expression in pyramidal neurons exhibited a dors